Ansamycin formulations and methods of use thereof

ABSTRACT

Provided herein, inter alia, are solid forms of geldanamycin analogs, pharmaceutical compositions comprising a geldanamycin analog and a crystallization inhibitor, methods of making and using such compositions. Additionally, provided are methods for the treatment of cancer, a neoplastic disease state and/or a hyperproliferative disorder, and methods of inhibiting Heat Shock Protein 90 (“Hsp90”).

RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) of the filingdate of U.S. Ser. No. 60/874,349 filed on Dec. 12, 2006, U.S. Ser. No.60/914,477 filed on Apr. 27, 2007, and 60/939,913 filed May 24, 2007,the entire disclosure of each of which is incorporated herein byreference.

FIELD

Provided herein are, inter alia, solid forms of geldanamycin analogs,pharmaceutical compositions comprising a geldanamycin analog and acrystallization inhibitor, and methods of making and using suchcompositions. In some embodiments, provided are methods for thetreatment of cancer and/or a hyperproliferative disorder, and methods ofinhibiting Heat Shock Protein 90 (“Hsp90”).

BACKGROUND

Hsp90 is an abundant protein which has a role in cell viability andwhich exhibits dual chaperone functions (J. Cell Biol. (2001)154:267-273, Trends Biochem. Sci. (1999) 24:136-141). It plays a role inthe cellular stress-response by interacting with many proteins aftertheir native conformations have been altered by various environmentalstresses, such as heat shock, ensuring adequate protein-folding andpreventing non-specific aggregation (Pharmacological Rev. (1998)50:493-513). Recent results suggest that Hsp90 may also play a role inbuffering against the effects of mutation, presumably by correctinginappropriate folding of mutant proteins (Nature (1998) 396:336-342).Hsp90 also has regulatory roles under normal physiological conditionsand is responsible for the conformational stability and maturation of anumber of specific client proteins (see. Expert. Opin. Biol Ther. (2002)2(1): 3-24).

Hsp90 antagonists are currently being explored in a large number ofbiological contexts where a therapeutic effect can be obtained for acondition or disorder by inhibiting one or more aspects of Hsp90activity.

Geldanamycin is a macrocyclic lactam that is a member of thebenzoquinone-containing ansamycin family of natural products.Geldanamycin's nanomolar potency and apparent selectivity for killingtumor cells, as well as the discovery that its primary target inmammalian cells is Hsp90, has stimulated interest in its development asan anti-cancer drug. However, its extremely low solubility and theassociation of hepatotoxicity with the administration of geldanamycinhave led to difficulties in developing an approvable agent fortherapeutic applications. In particular, geldanamycin has poor watersolubility, making it difficult to deliver in therapeutically effectivedoses.

More recently, attention has focused on 17-amino derivatives ofgeldanamycin (“geldanamycin analogs”), in particular 17-AAG, showingreduced hepatotoxicity while maintaining Hsp90 binding. See U.S. Pat.Nos. 4,261,989; 5,387,584; and 5,932,566. Like geldanamycin, these17-amino derivatives have very limited aqueous solubility. Consequently,there is an unmet need to develop additional pharmaceutical compositionsof geldanamycin analogs, such as 17-AG and 17-AAG, and solid formsthereof.

SUMMARY

In one embodiment, provided herein are solid forms of geldanamycinanalogs, which are useful as Hsp90 antagonists. Also provided herein,among other things, are pharmaceutical compositions comprisinggeldanamycin analogs, methods for making such compositions havingenhanced bioavailability, methods of using gelanamycin analogs for thetreatment of cancer and/or a hyperproliferative disorder, and methods ofinhibiting Hsp90. It has been discovered herein that mixtures ofgeldanamycin analogs and crystallization inhibitors dramatically improvethe bioavailability of geldanamycin analogs. Examples of formulationsthat achieve this improvement include, but are not limited to, soliddispersions, solid molecular dispersions, and physical blends of thecomponents. In some embodiments, a geldanamycin analog is present in anamorphous state, a microcrystalline state, a nanocrystalline state, orany combination thereof.

In certain embodiments, pharmaceutical compositions containing a soliddispersion of a geldanamycin analog and at least one crystallizationinhibitor are provided wherein the geldanamycin analog is present insubstantially amorphous form. In other embodiments, a method for thepreparation of amorphous geldanamycin analogs is provided. One methodfor producing a solid molecular dispersion of amorphous geldanamycinanalogs provided herein involves solvent spray drying. Other techniquesthat can be used to prepare solid molecular dispersions of amorphousgeldanamycin analogs include, without limitation: (1) milling; (2)extrusion; (3) melt processes, including high melt-congeal processes andmelt-congeal processes; (4) solvent modified fusion; (5) solventprocesses, including spray coating, lyophilization, solvent evaporation(e.g., rotary evaporation) and spray-drying; and (6) non-solventprecipitation.

In another embodiment, provided are amorphous geldanamycin analogs thatcan exist within a solid amorphous dispersion as a pure phase, as amolecular dispersion of geldanamycin analog homogeneously distributedthroughout a crystallization inhibitor or any combination of thesestates or those states that lie intermediate between them. In someembodiments, a dispersion is substantially homogeneous such that anamorphous geldanamycin analog is dispersed uniformly throughout thedispersion or formulation.

In yet another embodiment, provided are geldanamycin analogs that existin a variety of solid forms. In certain embodiments, 17-AG exists inmore than one polymorphic form. Provided are 17-AG compositions thatinclude such forms, whether in a pure polymorphic state or admixed withany other material, including for example, another polymorphic form of17-AG.

BRIEF DESCRIPTION OF THE DRAWING

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts an XRPD pattern for amorphous 17-AG.

FIG. 2 depicts an XRPD pattern for Form I of 17-AG.

FIG. 3 depicts a DSC pattern for Form I of 17-AG.

FIG. 4 depicts an ¹HNMR spectra for Form I of 17-AG.

FIG. 5 depicts an XRPD pattern for Form II of 17-AG.

FIG. 6 depicts an XRPD pattern for Form III of 17-AG.

FIG. 7 depicts an XRPD pattern for EtOAc Solvate of 17-AG.

FIG. 8 depicts a DSC pattern for EtOAc Solvate of 17-AG.

FIG. 9 an ¹HNMR spectra for EtOAc Solvate of 17-AG demonstrating ratioof 17-AG to ethyl acetate.

FIG. 10 a depicts a graph of 17-AG concentration levels, plotted as afunction of ng/ml versus time (hours), demonstrating higher relativebioavailability with the administration of amorphous 17-AG as comparedto crystalline 17-AG in male beagle dogs in: (i) an uncoated HPMCcapsule containing 17-AG (12% load) in a PVP solid dispersionformulation (Session 1); (ii) an uncoated HPMC capsule containing acrystalline 17-AG (Session 3); and (iii) a coated HPMC capsulecontaining 17-AG (12% load) in a PVP solid dispersion formulation(Session 4).

FIG. 10 b depicts a summary table of PK parameters for FIG. 10 a.

FIG. 11 a depicts a graph of 17-AG concentration levels, plotted as afunction of ng/ml versus time (hours), demonstrating higher relativebioavailability with the administration of amorphous 17-AG as comparedto crystalline 17-AG in female beagle dogs in: (i) an uncoated HPMCcapsule containing 17-AG (12% load) in a PVP solid dispersionformulation (Session 1); (ii) an uncoated HPMC capsule containing acrystalline 17-AG (Session 3); and (iii) a coated HPMC capsulecontaining 17-AG (12% load) in a PVP solid dispersion formulation(Session 4).

FIG. 11 b depicts a summary table of PK parameters for FIG. 11 a.

FIG. 12 depicts a DSC scan of 17-AG (12% load) in a PVP K-30 soliddispersion formulation made using lyophilization from t-BuOH/water(3:1).

FIG. 13 depicts a graph showing results from an in vitro dissolutionstudy of various 17-AG/polymer dispersions, plotted as a function ofmg/ml versus time (minutes).

FIG. 14 depicts a graph of 17-AG concentration levels, plotted as afunction of ng/ml versus time (hours), demonstrating relativebioavailability of 17-AG in female beagle dogs using: solid dispersionformulations made by two different methods, plotted as a function ofng/ml versus time (hours): (FIG. 14 a) an uncoated HPMC capsulecontaining 17-AG (20% load) in a PVP solid amorphous dispersionformulation made by rotary evaporation; and (FIG. 14 b) an uncoated HPMCcapsule containing 17-AG (20% load) in a PVP solid amorphous dispersionformulation made by spray drying.

FIG. 14 c depicts a summary table of the data in FIG. 14 a and FIG. 14b.

FIG. 15 depicts an in vitro dissolution study in SIF of amorphousdispersions of a series of ansamycin analogs generated from PVPutilizing rotary evaporation, plotted as a function of mg/ml versus time(minutes).

FIG. 16 depicts an XRPD pattern for an amorphous dispersion of 17-AGplus PVP (20% in K-30) made by rotary evaporation.

FIG. 17 depicts an XRPD pattern for an amorphous dispersion of 17-AGplus PVP spiked with 0.1% crystalline Form I.

FIG. 18 depicts an XRPD pattern for an amorphous dispersion of 17-AGplus PVP spiked with 1% crystalline Form I.

FIG. 19 depicts an XRPD pattern for an amorphous dispersion of 17-AGplus PVP spiked with spiked with 5% crystalline Form I.

FIG. 20 depicts an XRPD pattern for an amorphous dispersion of 17-AGplus PVP spiked with spiked with 10% crystalline Form I.

FIG. 21 depicts a graph showing results from an in vitro dissolutionstudy of a 17-AG/PVP dispersion containing varying amounts of Form II7-AG (0%, 1% and 10%), plotted as a function of mg/ml versus time(minutes), demonstrating the effect of varying amounts of Form I on thestability of supersaturated solutions.

FIG. 22 depicts a graph showing results from an in vitro dissolutionstudy of a 17-AG/PVP dispersion containing varying amounts of Form II17-AG (0%, 1% and 10%), plotted as a function of mg/ml versus time(minutes), demonstrating the effect of varying amounts of Form II on thestability of supersaturated solutions.

FIG. 23 depicts a graph showing results from an in vitro dissolutionstudy of a 17-AG/PVP dispersion containing varying amounts of Form III17-AG (0%, 1% and 10%), plotted as a function of mg/ml versus time(minutes), demonstrating the effect of varying amounts of Form III onthe stability of supersaturated solutions.

FIG. 24 depicts a graph showing the dissolution profile of 17-AG fromdifferent tablets and capsule (average values, in-vitro dissolution inSIF), demonstrating different dissolution/release profiles are possiblewith tablets of varying composition.

FIG. 25 on the left, depicts a three-dimensional bar graph of anin-vitro dissolution study using 0.5 mg/ml 17-AG in various SIFsolutions containing 0%, 0.5%, 1.5% and 5% PVP (50 mg/ml 17-AG in DMSOdiluted 1:100 into SIF); and on the right, depicts a three-dimensionalbar graph of an in-vitro solubility study using 1.0 mg/ml 17-AG invarious SIF solutions containing 0%, 0.5%, 1.5% and 5% PVP (100 mg/ml17-AG in DMSO diluted 1:100 into SIF); together demonstrating thatvaried amounts of PVP achieve supersaturation levels of 17-AG andstabilizes the supersaturated solutions by preventingnucleation/precipitation of 17-AG.

FIG. 26 depicts a three-dimensional bar graph demonstrating that variedamounts of PVP in SIF will change the degree of supersaturation of 17-AGin SIF, i.e., higher amounts of PVP result in higher supersaturatedlevels of 17-AG.

FIG. 27 depicts a graph of the equilibrium solubility of 17-AG in SIFcontaining varying amounts of PVP K-30 (0%, 0.5%, 1%, 2.5% and 5%),plotted as a function of mg/ml versus time, demonstrating that addingPVP increases the equilibrium solubility of 17-AG in simulatedintestinal fluid (SIF).

FIG. 28 a depicts a graph demonstrating effects of varying loads of17-AG (12%, 20% and 30% loads (w/w) in PVP K-30) on plasma levelconcentration of 17-AG in female beagle dogs, plotted as a function ofng/ml versus time (minutes). FIG. 28 b is a summary table of the data inFIG. 28 a.

FIG. 29 a depicts a graph demonstrating effects of varying loads of17-AG (12%, 20% and 30% loads (w/w) in PVP K-30) on plasma levelconcentration of 17-AG in male beagle dogs, plotted as a function ofng/ml versus time (minutes).

FIG. 29 b depicts a summary table of the data in FIG. 29 a.

FIG. 30 a depicts a graph of 17-AG dog plasma level concentration afteradministration of an uncoated HPMC capsule containing a physical blendof an EtOAc Solvate of 17-AG and lactose, without a crystallizationinhibitor present, plotted as a function of ng/ml versus time (hours).

FIG. 30 b depicts a graph of 17-AG dog plasma level concentration afteradministration of an uncoated HPMC capsule containing a physical blendof an EtOAc Solvate of 17-AG and a crystallization inhibitor (PVP),plotted as a function of ng/ml versus time (hours) demonstrating thatadding a crystallization inhibitor leads to increased plasma levelconcentration.

FIG. 30 c depicts a summary table of the PK parameters for FIG. 30 a andFIG. 30 b.

FIG. 31 a depicts a graph of 17-AG dog plasma level concentration afteradministration of an uncoated HPMC capsule containing amorphous 17-AGand lactose, without a crystallization inhibitor present, plotted as afunction of ng/ml versus time (hours), demonstrating that even when nocrystallization inhibitor is present, the plasma level concentration ishigh, relative to crystalline 17-AG.

FIG. 31 b depicts a graph of 17-AG dog plasma level concentration afteradministration of an uncoated HPMC capsule containing amorphous 17-AGand a crystallization inhibitor (PVP), plotted as a function of ng/mlversus time (hours), demonstrating that adding a crystallizationinhibitor leads to increased plasma level concentration.

FIG. 31 c depicts a summary table of the PK parameters for FIG. 31 a andFIG. 31 b.

FIG. 32 depicts a graph of dog plasma level concentration afteradministration of 17-AG as a solution (85% propylene glycol, 5% ethanoland 10% DMSO) via oral gavage, without a crystallization inhibitorpresent, plotted as a function of ng/ml versus time (hours).

FIG. 33 depicts a graph of dog plasma level concentration afteradministration of 17-AG as a solution (85% propylene glycol, 5% ethanoland 10% PVP) via oral gavage containing a crystallization inhibitor(PVP), plotted as a function of ng/ml versus time (hours), demonstratingthat solutions as well as solid dispersions are effective formulationsfor oral administration.

FIG. 34 depicts a graph of dog plasma level concentration afteradministration of 17-AG as a solution (20% polyethyleneglycol-hydroxystearate, 5% DMSO in normal saline) via oral gavage,containing a crystallization inhibitor (PEG-HS), plotted as a functionof ng/ml versus time (hours).

FIG. 35 depicts a graph of dog plasma level concentration afteradministration of 17-AG as a solution (20% polyethyleneglycol-hydroxystearate, 5% DMSO, 10% PVP in normal saline) via oralgavage, containing a crystallization inhibitor (PEG-HS and PVP), plottedas a function of ng/ml versus time (hours).

FIG. 36 depicts a graph of female dog plasma level concentration afteradministration if 17-AG as a solution (non-ionic 2% Tween-80, 5% DMSO insterile water for injection) via oral gavage, containing acrystallization inhibitor (non-ionic Tween-80), plotted as a function ofng/ml versus time (hours).

FIG. 37 depicts a graph of dog plasma level concentration afteradministration of 17-AG as a solution (non-ionic 2% Tween-80, 5% DMSO,10% PVP in sterile water for injection) via oral gavage, containing acrystallization inhibitor (PVP and non-ionic Tween-80), plotted as afunction of ng/ml versus time (hours).

FIG. 38 depicts a graph of a relative in vitro dissolution study (SIF at37° C.) of amorphous 17-AG (12% load) in solid dispersions using variousgrades of PVP (K-15, K-30 and K-90) at 37° C., plotted as a function of% of Target (2 mg/ml) versus time (minutes). The trend that PVP K-90resulted in lower supersaturated levels of 17-AG than PVP K-15 or PVPK-30 grades was consistent regardless of the 17-AG load.

FIG. 39 depicts a graph of a relative in vitro dissolution study (SIF at37° C.) of amorphous 17-AG plus PVP dispersions using varying loadlevels of 17-AG (12%, 20%, 30% and 50%). The trend that supersaturatedlevels of 17-AG was inversely correlated to load (i.e. dispersions withhigher load levels of 17-AG and lower levels of crystallizationinhibitor, PVP, had lower supersaturated levels of 17-AG) was consistentregardless of the PVP grade.

FIG. 40 a depicts a graph of plasma concentration in female beagle dogsafter oral dosing (10 mg/kg) of 17-AG (20% load) plus PVP in a soliddispersion formulation [both small (<50 μM) and large (>800 μM) particlesizes], plotted as a function of ng/ml versus time (minutes)demonstrating that particle size does not greatly affect in-vivoexposure.

FIG. 40 b depicts a summary table of the PK parameters for FIG. 40 a.

FIG. 40 c depicts a summary table of the PK parameters for a graph ofplasma concentration in male beagle dogs after oral dosing (10 mg/kg) of17-AG (20% load) plus PVP in a solid dispersion formulation [both small(less than 50 microns) and large (greater than 800 microns) particlesizes], plotted as a function of ng/ml versus time (minutes).

FIG. 41 a depicts a graph of 17-AG (12% load) plasma concentration infemale beagle dogs after oral dosing (15 mg/kg) of amorphous 17-AGdispersions using various grades of PVP (K-15, K30 and K-90), plotted asa function of ng/ml versus time (hours) demonstrating a trend. From thedata, PVP grade K-30 provides greater exposure than PVP K-15 whichprovides greater exposure than PVP K-90.

FIG. 41 b depicts a summary table of the PK parameters for FIG. 41 a.

FIG. 42 a depicts a graph of 17-AG (12% load) plasma concentration inmale beagle dogs after oral dosing (15 mg/kg) of amorphous 17-AGdispersions using various grades of PVP (K-15, K-30 and K-90), plottedas a function of ng/ml versus time (hours). Similar to the data fromfemale dog dosing, PVP grade K-30 provides greater exposure than PVPK-15 which provides greater exposure than PVP K-90.

FIG. 42 b depicts a summary table of the PK parameters for FIG. 42 a.

FIG. 43 depicts a graph of plasma levels achieved after a single capsuledose of an amorphous dispersion (12% 17-AAG, with a crystallizationinhibitor PVP in uncoated HPMC capsule) in beagle dogs, demonstratingthat good in-vivo exposure can be achieved in dosing amorphousdispersions of ansamycin analogs other than 17-AG.

FIG. 44 depicts exemplary images acquired using a polarized lightmicroscope: (A) 4× transmitted light image of solid state 17AG/PVPamorphous dispersion; (B) 10× polarized light image of 17AG/PVPamorphous dispersion in water; (C) 10× polarized light image of 0.01%spike of crystalline Form I into a 17-AG/PVP amorphous dispersion,dissolved in water; (D) 10× polarized light image of 0.1% spike ofcrystalline Form I into a 17-AG/PVP amorphous dispersion, dissolved inwater; and (E) 10× polarized light image of 1.0% spike of crystallineForm I into a 17-AG/PVP amorphous dispersion, dissolved in water.

FIG. 45 depicts a transmitted light micrograph of the 17-AG/PVPdispersion made by lyophilization from 3:1 t-BuOH/water, dissolved inwater.

FIG. 46 depicts exemplary photo images of a suspension and an emulsion:(A) 2% 17-AG suspension in 1% Carboxymethylcellulose; and (B) 2 mg/ml17-AG in 10% PGHS, 2.5% DMSO, 5% Tween-80, 50% olive oil in NS.

FIG. 47 depicts a graph showing tumor volume as a function of time(days) utilizing a mouse xenograph model H1975 when dosed using asolution of 17-AG in 20% PG-HS, 5% DMSO and 75% normal saline.

FIG. 48 depicts a graph showing tumor volume as a function of time(days) utilizing a mouse xenograph model H1650 when dosed using asolution of 17-AG in 15% PVP, 5% ethanol and 80% propylene glycol.

DETAILED DESCRIPTION (1) Definitions and Abbreviations

The definitions of terms used herein are meant to incorporate thepresent state-of-the-art definitions recognized for each term in thechemical and pharmaceutical fields. Where appropriate, exemplificationis provided. The definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

Where stereochemistry is not specifically indicated, all stereoisomersof the inventive compounds provided herein are included within the scopeof this disclosure, as pure isomers as well as mixtures thereof. Unlessotherwise indicated, individual enantiomers, diastereomers, geometricalisomers, and combinations and mixtures thereof are all encompassed bythe present disclosure. Polymorphic crystalline forms and solvates arealso encompassed within the scope of this disclosure.

The term “acylamino” and “acylamine” refers to a moiety that may berepresented by the general formula:

wherein each of R50 and R51 independently represent a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R61; wherein R61 represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8; or R50 and R51, taken togetherwith the N atom to which they are attached complete a heterocycle havingfrom 4 to 8 atoms in the ring structure.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In certain embodiments, a straightchain or branched chain alkyl has 30 or fewer carbon atoms in itsbackbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branched chain),20 or fewer. In some embodiments, certain cycloalkyls have from 3-10carbon atoms. In some embodiments, an alkyl group contains 1-10 carbonatoms as its backbone, and may be substituted. In some embodiments,certain cycloalkyls have from 3-10 carbon atoms in their ring structure,and others have 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to aboutten carbons, alternatively from one to about six carbon atoms in itsbackbone structure. In some embodiments, “lower alkenyl” and “loweralkynyl” have similar chain lengths from two to about ten carbons,alternatively from two to about six carbon atoms in its backbonestructure.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “aralkyl” is art-recognized and refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup). Benzyl, p-methoxybenzyl, and phenylethyl are examples of anaralkyl.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.Alkenyl and alkynyl groups may be substituted with the same groups thatare suitable as substituents on alkyl groups, to the extent permitted bythe available valences. In certain embodiments, alkenyl and alkynylgroups contain 2-10 carbons in the backbone structure.

The terms “alkoxyl” or “alkoxy” refers to an alkyl group, as definedherein, having an oxygen radical attached thereto. In one embodiment,alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and thelike. The alkyl portion of an alkoxy group is sized like the alkylgroups, and can be substituted by the same groups that are suitable assubstituents on alkyl groups, to the extent permitted by the availablevalences.

The term “amido” and “amide” are art recognized as an amino-substitutedcarbonyl and includes a moiety that may be represented by the generalformula:

wherein R50 and R51 are as defined herein.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50, R51 and R52 each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R61, or R50 and R51, taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R61 represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In other embodiments, R50 and R51(and optionally R52) each independently represent a hydrogen, an alkyl,an alkenyl, or —(CH₂)_(m)—R61. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R50 and R51 is an alkylgroup.

The term “aralkyl” as used herein, whether alone or as part of a groupname such as, for example, aralkyloxy, refers to an alkyl group asdescribed herein substituted with an aryl group as described herein(e.g., an aromatic or heteroaromatic group). The aryl portion of eacharalkyl group may be optionally substituted. In one embodiment, aralkylgroups include, for example, groups of general formula Ar—(CH₂)_(t)—,where Ar represents an aromatic or heteroaromatic ring and t is aninteger from 1-6.

The term “aryl” as used herein, whether alone or as part of another nameas in ‘aryloxy’, refers to 5-, 6- and 7-membered single-ring aromaticgroups that may include from zero to four heteroatoms selected from N, Oand S, for example, benzene, naphthalene, anthracene, pyrene, pyrrole,furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles” or “heteroaromatics.” The aromatic ring may besubstituted at one or more ring positions with such substituents asdescribed herein, for example, halogen, azide, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,aromatic or heteroaromatic moieties, —CF₃, —CN, or the like. The term“aryl” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining rings(the rings are “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

As used herein, the term “benzoquinone ansamycin” (aka “geldanamycincompound”) means a macrocyclic lactam ring system containing: (a) oneamide bond; and (b) a benzoquinone moiety, wherein said benzoquinonemoiety bears 0-2 nitrogen substituents that are exo- to the macrocycliclactam ring system, and the benzoquinone moiety itself. Specificexamples of naturally-occurring benzoquinone ansamycins include, but arenot limited to, geldanamycin and herbimycin.

The phrase “characteristic XRPD peaks” or “characteristic set of peaks”means a single peak or set of peaks taken from an XRPD spectra thatdistinguish a polymorph from other known polymorphs of the same compoundidentified by comparison of XRPD patterns from different forms.

The term “crystallization inhibitor” means a pharmaceutically acceptableexcipient which substantially inhibits the conversion of a compound fromthe amorphous form to one or more crystalline forms in the solid stateor in solution. A crystallization inhibitor may also substantiallyinhibit crystal growth in the gastrointestinal tract for long enough(e.g., about 1 to 6 hours) to allow for enhanced absorption of at least50%, over conventional delivery, of the compound into the bloodstream.

The term “geldanamycin analog” refers to a benzoquinone ansamycin otherthan geldanamycin, for example, 17-amino-geldanamycin (17-AG),17-allylamino-17-demethoxygeldanamycin (17-AAG) or17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG).

The term “heterocycloalkyl” refers to cycloalkyl groups as describedherein, wherein at least one carbon atom of the alkyl or cycloalkylportion is replaced by a heteroatom selected from N, O and S.

The term “heteroatom” is art-recognized and refers to an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The terms “heterocyclyl”, “heteroaryl”, “heterocyclic ring” or“heterocyclic group” are art-recognized and refer to 3- to about10-membered ring structures, alternatively 3- to about 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles mayalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringmay be substituted at one or more positions with such substituents asdescribed herein, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The term “Hsp90 mediated disorder” or “disorder mediated by cellsexpressing Hsp90” refers to pathological and disease conditions in whichHsp90 plays a role. Such roles can be directly related to thepathological condition or can be indirectly related to the condition.The common feature to this class of conditions is that the condition canbe ameliorated by inhibiting the activity, function, or association withother proteins of Hsp90. Particular exemplary Hsp90 mediated disordersare discussed, infra.

As used herein, the term “isolated” in connection with a compoundprovided herein means the compound is not in a cell or organism and thecompound is separated from some or all of the components that typicallyaccompany it in nature.

The term “molecular dispersion” as used herein refers to a type of soliddispersion wherein one component is dispersed throughout anothercomponent such that the system is chemically and physically uniform andhomogeneous throughout. These systems are substantially free of activeingredients in their crystalline or microcrystalline state as evidencedby thermal analysis (e.g., differential scanning calorimetry),diffractive (e.g., X-ray diffraction), or imaging (polarized lightmicroscopy) techniques.

The term “pharmaceutically acceptable salt” or “salt” refers to a saltof one or more compounds. Suitable pharmaceutically acceptable salts ofcompounds include acid addition salts which may, for example, be formedby mixing a solution of the compound with a solution of apharmaceutically acceptable acid, such as hydrochloric acid, hydrobromicacid, sulfuric acid, fumaric acid, maleic acid, succinic acid, benzoicacid, acetic acid, citric acid, tartaric acid, phosphoric acid, carbonicacid, or the like. Where the compounds carry one or more acidicmoieties, pharmaceutically acceptable salts may be formed by treatmentof a solution of the compound with a solution of a pharmaceuticallyacceptable base, such as lithium hydroxide, sodium hydroxide, potassiumhydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodiumcarbonate, potassium carbonate, ammonia, alkylamines, or the like.

The term “pharmaceutically acceptable carrier” includes any and allsolvents, diluents, or other liquid vehicle, dispersion or suspensionaids, surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. Remington'sPharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutical compositions and known techniques for thepreparation thereof. Except insofar as any conventional carrier mediumis incompatible with the compounds of provided herein, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatine; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil, sesame oil; olive oil; corn oil and soybean oil; glycols; such aspropylene glycol; esters such as ethyl oleate and ethyl laurate; agar;buffering agents such as magnesium hydroxide and aluminum hydroxide;alginic acid; pyrogenfree water; isotonic saline; Ringer's solution;ethyl alcohol, and phosphate buffer solutions, as well as othernon-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The terms “polycyclyl” or “polycyclic group” are art-recognized andrefer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The phrase “protecting group”, as used herein means that a particularfunctional moiety, e.g. O, S, or N, is temporarily blocked so that areaction can be carried out selectively at another reactive site in amultifunctional compound. In certain embodiments, a protecting groupreacts selectively in good yield to give a protected substrate that isstable to the projected reactions; the protecting group must beselectively removed in good yield by readily available, preferablynontoxic reagents that do not attack the other functional groups; theprotecting group may form an easily separable derivative (without thegeneration of new stereogenic centers); and the protecting group has aminimum of additional functionality to avoid further sites of reaction.As detailed herein, oxygen, sulfur, nitrogen and carbon protectinggroups may be utilized. For example, in certain embodiments, as detailedherein, certain exemplary protecting groups include esters of carboxylicacids, silyl ethers of alcohols, and acetals and ketals of aldehydes andketones, respectively. Certain other exemplary protecting groups aredetailed herein, however, it will be appreciated that the presentinvention is not intended to be limited to these protecting groups;rather, a variety of additional equivalent protecting groups can bereadily identified using the above criteria and utilized in the presentinvention. Additionally, a variety of protecting groups are described in“Protective Groups in Organic Synthesis” Third Ed. Greene, T. W. andWuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entirecontents of which are hereby incorporated by reference.

The term “polymorph” refers to different crystal structures achieved bya particular chemical entity. Specifically, polymorphs occur when aparticular chemical compound can crystallize with more than onestructural arrangement.

The term “solvate” refers to a crystal form where a stoichiometric ornon-stoichiometric amount of solvent, or mixture of solvents, isincorporated into the crystal structure.

The term “subject” as used herein, refers to an animal, typically amammal or a human, that will be or has been the object of treatment,observation, and/or experiment. When the term is used in conjunctionwith administration of a compound or drug, then the subject has been theobject of treatment, observation, and/or administration of the compoundor drug.

The term “substantially amorphous” when used to describe a compositiondisclosed herein means that the majority of the compound present in acomposition is present in amorphous form and that the composition hasless than about 20% crystalline compound, less than about 15%crystalline compound, less than about 10% crystalline compound, lessthan about 5% crystalline compound, less than about 3% crystallinecompound, or less than about 1% crystalline compound, less than about0.1% crystalline compound, or less than about 0.01% crystallinecompound. In some embodiments of the present invention, the compoundpresent in a composition contains no detectable crystalline material.When the term “substantially amorphous” is used to describe a compounddisclosed herein it means that the majority of the compound is presentin amorphous form and the compound has less than about 20% crystallinecontent, less than about 15% crystalline content, less than about 10%crystalline content, less than about 5% crystalline content, less thanabout 3% crystalline content, or less than about 1% crystalline content,less than about 0.1% crystalline content, or les than about 0.01%crystalline content. In some embodiments of the present invention, thecompound present in a composition contains no detectable crystallinematerial.

The term “substantially free of”, when used to describe a material orcompound, means that the material or compound lacks a significant ordetectable amount of a designated substance. In some embodiments, thedesignated substance is present at a level not more than about 1%, 2%,3%, 4% or 5% (w/w or v/v) of the material or compound. For example, apreparation of a particular geldanamycin analog is “substantially freeof” other geldanamycin analogs if it contains less than about 1%, 2%,3%, 4% or 5% (w/w or v/v) of any geldanamycin analog other than theparticular geldanamycin analog designated. Similarly, in someembodiments of the present invention, a preparation of amorphousgeldanamycin is “substantially free of” crystalline geldanamycin if itcontains less than about 1%, 2%, 3%, 4% or 5% (w/w or v/v) crystallinegeldanamycin. In some embodiments of the present invention, thepreparation of amorphous geldanamycin contains no detectable crystallinegeldanamycin. Similarly, in some embodiments of the present invention apreparation of amorphous 17-AG is “substantially free of” crystalline17-AG if it contains less than about 1%, 5%, 10% or 15% (w/w or v/v)crystalline 17-AG. Similarly, in some embodiments of the presentinvention a preparation of amorphous 17-AAG is “substantially free of”crystalline 17-AAG if it contains less than about 1%, 5%, 10% or 15%(w/w or v/v) crystalline 17-AAG. Similarly, in some embodiments of thepresent invention, a preparation of an EtOAc solvate is “substantiallyfree of” other solid forms of 17-AG if it contains less than about 1%,5%, 10% or 15% (w/w or v/v) of any solid form other than the solid formdesignated. Similarly, in some embodiments of the present invention, apreparation of an EtOAc solvate is “substantially free of” other solidforms of 17-AAG if it contains less than about 1%, 5%, 10% or 15% (w/wor v/v) of any solid form other than the solid form designated.

The phrase “substantially all” when used to describe XRPD peaks of acompound means that the XRPD of that compound includes at least about80% of the peaks when compared to a reference. For example, when an XRPDof a compound is said to include “substantially all” of the peaks in areference list, or all of the peaks in a reference XRPD, it means thatthe XRPD of that compound includes at least 80% of the peaks in thespecified reference. In other embodiments, the phrase “substantiallyall” means that the XRPD of that compound includes at least about 85,90, 95, 97, 98, or 99% of the peaks when compared to a reference.Additionally, one skilled in the art will appreciate throughout, thatXRPD peak intensities and relative intensities as listed herein maychange with varying particle size and other relevant variables.

The term “substantially homogeneous” means that the geldanamycin analogis dispersed evenly throughout the dispersion or formulation. Thus, aportion of a dispersion that is 10% by weight of the dispersion shouldcontain 8-12% or 9-11% by weight of the geldanamycin analog present inthe dispersion.

The term “substantially inhibit” as used herein means to reducesignificantly. For example, a crystallization inhibitor that inhibitsconversion of an amorphous compound to one or more crystalline forms ofthe compound in the solid state (e.g., that “substantially inhibits”that conversion if it reduces conversion to less than about 1%, to lessthan about 5%, to less than about 10%, to less than about 15%, to lessthan about 20%, or less than about 25% crystalline material) for aperiod of about 1 hour or greater, about 3 hours or greater, about 6hours or greater, about 12 hours or greater, about 1 day or greater,about 1 week or greater, about one month or greater, about 3 months orgreater, about 6 months or greater, or about one year or greater.

The term “substituted” refers to a chemical group, such as alkyl,cycloalkyl aryl, and the like, wherein at least one hydrogen is replacedwith a substituent as described herein, for example, halogen, azide,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino,nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone,aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties,—CF₃, —CN, or the like. The term “substituted” is also contemplated toinclude all permissible substituents of organic compounds. In a broadaspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnonaromatic substituents of organic compounds. Illustrative substituentsinclude, for example, those described herein above. The permissiblesubstituents may be one or more and the same or different forappropriate organic compounds. For purposes of this disclosure, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. This disclosure is not intendedto be limited in any manner by the permissible substituents of organiccompounds. In many embodiments, however, any single substituent hasfewer than the 100 total atoms.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The definition of each expression, e.g. alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

The term “sugar” as used herein refers to a natural or an unnaturalmonosaccharide, disaccharide, oligosaccharide, or polysaccharidecomprising one or more triose, tetrose, pentose, hexose, heptose,octose, and nonose saccharides. Sugars may include alditols resultingfrom reduction of the saccharide carbonyl group; aldonic acids resultingfrom oxidation of one or more terminal groups to carboxylic acids of thesaccharide; deoxy sugars resulting from replacement of one or morehydroxyl group(s) by a hydrogen in the saccharide; amino sugarsresulting from replacement of one or more hydroxyl group(s) by an aminogroup in the saccharide; thio sugars resulting from replacement of oneor more hydroxyl group(s) by a thiol group, or other analogous compoundsresulting from the replacement of, for example, one or more hydroxylgroup(s) by an acylamino group, a sulfate group, a phosphate group, orsimilar heteroatomic group; or any combination of the foregoingmodifications. The term sugar also includes analogs of these compounds(I.e., sugars that have been chemically modified by acylation,alkylation, and formation of glycosidic bonds by reaction of sugaralcohols with aldehydes or ketones, etc). Sugars may be present incyclic (oxiroses, oxetosesm furanoses, pyranoses, septanoses, octanoses,etc) form as hemiacetals, hemiketals, or lactones; or in acyclic form.The sacharides may be ketoses, aldoses, polyols and/or a mixture ofketoses, aldoses and polyols. Sugars may include, but are not limited toglycerol, polyvinylalcohol, propylene glycol, sorbitol, ribose,arabinose, xylose, lyxose, allose, altrose, mannose, mannitol, gulose,dextrose, idose, galactose, talose, glucose, fructose, dextrates,lactose, sucrose, starches (i.e., amylase and amylopectin), sodiumstarch glycolate, cellulose and cellulose derivativees (i.e.,methylcellulose, hydroxypropyl celluloe, hydroxyethyl cellulose,hydroxyethylmethyl cellulose, carboxymethyl cellulose, celluloseacetate, cellulose acetate phthalate, croscarmellose, hypomellose, andhydroxypropyl methyl cellulose), carrageenan, cyclodextrins, dextrin,polydextrose, and trehalose.

The term “supersaturated” means that a solution has a concentration ofdissolved solute that is higher than the concentration of that samesolute at equilibrium solubility in a given solvent at a giventemperature.

The phrase “therapeutically effective amount” as used herein, means anamount sufficient to elicit a desired biological or medicinal responsein a cell culture, tissue system, animal, or human. In some embodiments,the response includes alleviation and/or delay of onset of one or moresymptoms of the disease, condition, or disorder being treated.

The phrase “taken together form a bond,” when used to refer to twochemical groups means that, if the groups are attached to atoms that arenot otherwise directly bonded to each other, they represent a bondbetween the atoms to which they are attached. If the groups are on atomsthat are directly bonded to each other, they represent an additionalbond between those two atoms. Thus, for example, when R⁵ and R⁶ takentogether form a bond, the structure —CH(R⁵)—CH(R⁶)— represents—C(H)═C(H)—.

Certain compounds contained in compositions disclosed herein may existin particular geometric or stereoisomeric forms. Unless otherwiseindicated, the present disclosure contemplates all such compounds,including cis- and trans-isomers, R- and S-enantiomers, diastereomers,(D)-isomers, (L)-isomers, the racemic mixtures thereof, and othermixtures thereof, as falling within the scope of this disclosure.Additional asymmetric carbon atoms may be present in a substituent suchas an alkyl group. All such isomers, as well as mixtures thereof, areintended to be included in this disclosure.

(2) Solid Forms

Provided herein are geldanamycin analogs that can exist in a variety ofsolid forms. Such forms include neat crystal forms, known as polymorphs.Such solid forms also include solvates, hydrates, anhydrous forms andamorphous. Such solid forms of geldanamycin analogs are contemplated aswithin this disclosure. In certain embodiments, provided is ageldanamycin compound as a mixture of one or more different solid forms(e.g., polymorphs, solvates and amorphous geldanamycin analogs) of 17-AGand/or 17-AAG.

Provided herein is 17-AG as an amorphous solid, referred to herein asamorphous 17-AG, and substantially free of other geldanamycin analogs.In some embodiments, amorphous 17-AG is substantially free of othersolid forms of 17-AG. Amorphous solids are well known to one of ordinaryskill in the art and are typically prepared by such methods aslyophilization, melting, and precipitation from supercritical fluid,among others. Methods of preparing amorphous 17-AG are described in theExamples section, infra.

In some embodiments, amorphous 17-AG is characterized in that it has anXRPD pattern similar to that depicted in FIG. 1.

In certain embodiments, provided is substantially amorphous 17-AGsubstantially free of crystalline forms of 17-AG.

Provided herein are at least three polymorphic forms, referred to hereinas Form I, Form II and Form III, of 17-AG.

In certain embodiments, provided is Form I of 17-AG. In someembodiments, provided is Form I of 17-AG characterized in that it has apeak in its XRPD patterns at the specified peaks±about 0.3 degrees2-theta. As used herein, the term “about”, when used in reference to anydegree 2-theta value recited, refers to the stated value ±0.3 degree2-theta in accordance with the value's reported decimal place.

In certain embodiments, provided is Form I of 17-AG substantially freeof other geldanamycin analogs. In some embodiments, Form I of 17-AG issubstantially free of other solid forms of 17-AG. In some embodiments,Form I is characterized by representative peaks in its XRPD patternselected from those at about 6.2, 8.5, 13.6, 15.9, 16.9, 22.4, 23.4,26.3, 30.6, 31.7, 35.1 and 36.1 degrees 2-theta, and combinationsthereof. In some embodiments, Form I is characterized in that it has atleast one peak selected from those at about 6.2, 8.5 13.6 and 15.9degrees 2-theta. In some embodiments, Form I is characterized by atleast one representative peak in its XRPD pattern selected from those atabout 6.2, 8.5, 13.6 and 15.9, in combination with at least one otherpeak selected from those at about 6.2, 8.5, 13.6, 15.9, 16.9, 22.4,23.4, 26.3, 30.6, 31.7, 35.1 and 36.1 degrees 2-theta. In someembodiments, Form I of 17-AG is characterized in that it hassubstantially all peaks in its XRPD pattern shown in FIG. 2.

In some embodiments, Form I is characterized in that it has a DSCpattern similar to that depicted in FIG. 3. A representative ¹HNMRspectra for Form I is depicted in FIG. 4.

In certain embodiments, provided is Form II of 17-AG substantially freeof other geldanamycin analogs. In some embodiments, Form II of 17-AG issubstantially free of other solid forms of 17-AG. In some embodiments,Form II is characterized by representative peaks in its XRPD patternselected from those at about 9.5, 10.1, 12.5, 15.1, 16.1, 16.8, 19.8,20.7, 21.5, 22.4, 25.1, 25.8, 29.5 and 30.5 degrees 2-theta, andcombinations thereof. In some embodiments, Form II is characterized inthat it has at least one peak selected from those at about 12.5, 15.1,20.7, 22.4 and 25.0 degrees 2-theta. In some embodiments, Form II ischaracterized by at least one representative peak in its XRPD patternselected from those at about 12.5, 15.1, 20.7, 22.4 and 25.0 incombination with at least one other peak selected from those at about9.5, 10.1, 12.5, 15.1, 16.1, 16.8, 19.8, 20.7, 21.5, 22.4, 25.1, 25.8,29.5 and 30.5 degrees 2-theta. In some embodiments, Form II ischaracterized by its XRPD peaks substantially as shown in FIG. 5.

In certain embodiments, provided is Form III of 17-AG substantially freeof other geldanamycin analogs. In some embodiments, Form III of 17-AG issubstantially free of other solid forms of 17-AG. In some embodiments,Form III is characterized by representative peaks in its XRPD patternselected from those at about 8.4, 9.3, 10.9, 11.6, 13.6, 13.9, 15.7,16.3, 17.1, 18.3, 18.6, 19.9, 21.0, 22.0, 24.3, 25.8, 28.2, 29.2, and30.8 degrees 2-theta, and combinations thereof. In some embodiments,Form III is characterized in that it has at least one peak selected fromthose at about 18.3, 21.0 and 24.3 degrees 2-theta. In some embodiments,Form III is characterized by at least one representative peak in itsXRPD pattern selected from those at about 18.3, 21.0 and 24.3, incombination with at least one other peak selected from those at about8.4, 9.3, 10.9, 11.6, 13.6, 13.9, 15.7, 16.3, 17.1, 18.3, 18.6, 19.9,21.0, 22.0, 24.3, 25.8, 28.2, 29.2 degrees 2-theta. In some embodiments,Form III is characterized by its XRPD peaks substantially as shown inFIG. 6.

Also provided is at least one solvate form, referred to herein as EtOAcSolvate, of 17-AG.

In certain embodiments, provided is an ethyl acetate solvate of 17-AGsubstantially free of other geldanamycin analogs. In some embodiments,the EtOAc Solvate of 17-AG is substantially free of other solid forms of17-AG. In some embodiments, the EtOAc Solvate is characterized byrepresentative peaks in its XRPD pattern selected from those at about6.2, 8.2, 12.6, 14.5, 15.9, 16.8, 17.5, 22.3, 23.3 and 25.3 degrees2-theta, and combinations thereof. In some embodiments, the EtOAcSolvate is characterized in that it has at least one peak selected fromthose at about 8.2, 15.9 and 22.3 degrees 2-theta. In some embodiments,the ethyl acetate solvate is characterized by at least onerepresentative peak in its XRPD pattern selected from those at about8.2, 15.9 and 22.3, in combination with at least one other peak selectedfrom those at about 6.2, 8.2, 12.6, 14.5, 15.9, 16.8, 17.5, 22.3, 23.3and 25.3 degrees 2-theta. In some embodiments, the EtOAc Solvate ischaracterized by its XRPD peaks substantially as shown in FIG. 7.

In some embodiments, the EtOAc solvate is characterized in that it has aDSC pattern similar to that depicted in FIG. 8. In some embodiment, theDSC shows an endothermic transition at about 96° C., consistent with adesolvation event. In some embodiments, the DSC shows an endothermictransition (melting point onset) at about 276° C.

In some embodiments, the EtOAc Solvate is characterized by its ¹HNMRpeaks substantially as shown in FIG. 9 below, showing that there is acomplex between 17-AG and ethyl acetate in approximate ratio of 2:1.

In another embodiment, also provided herein is 17-AAG can exist as anamorphous solid, referred to herein as amorphous 17-AAG, that issubstantially free of other geldanamycin analogs. In some embodiments,amorphous 17-AAG is substantially free of other solid forms of 17-AAG.Amorphous solids are well known to one of ordinary skill in the art andare typically prepared by such methods as lyophilization, melting, andprecipitation from supercritical fluid, among others. Methods ofpreparing amorphous 17-AAG are described in the Examples section, infra.

In certain embodiments, provided is substantially amorphous 17-AAGsubstantially free of other crystalline forms of 17-AAG.

In some embodiments, provided is a composition comprising amorphous17-AAG and at least one crystalline form of 17-AAG. Such crystallineforms of 17-AAG include neat crystal forms, solvates and hydrates asdescribed herein or other crystalline forms of 17-AAG that may resultfrom the preparation of, and/or isolation of, amorphous 17-AAG. Incertain embodiments, provided is a composition comprising amorphous17-AAG and at least one crystalline form of 17-AAG as described herein.In some embodiments, provided is a composition comprising amorphous17-AAG and at least one crystalline form of 17-AAG.

(3) Pharmaceutical Compositions

It is art-recognized that geldanamycin and other benzoquinone ansamycincompounds (including, for example, 17-AG and 17-AAG) are poorly solublein water, and thus are not suitable for oral administration due to poorbioavailability. Provided herein are pharmaceutical compositions of suchcompounds that can be administered orally if they, for example, aredelivered in an amorphous form (and/or in the presence of acrystallization inhibitor). In one embodiment, provided are oralformulations of benzoquinone ansamycin compounds, such as 17-AG or17-AAG, which oral formulations comprise amorphous compound in a solidor liquid composition, optionally also including a crystallizationinhibitor. In some embodiments, compositions that contain a mixture ofan amorphous geldanamycin analog and a crystallization inhibitor,resulted in a surprising finding that the bioavailability of amorphousgeldanamycin analogs are dramatically improved and are therefore usefulfor oral administration.

Without wishing to be bound by any particular theory, we propose thatone mechanism that might contribute to the improved bioavailability ofinventive oral formulations provided herein might be reducedrecrystallization of compound as it is released from the formulation inthe gastrointestinal tract. That is, if absorption of a compound is slowas it is released from a delivered formulation, then the possibilityexists that a supersaturated solution is generated in thegastrointestinal tract, potentially resulting in crystallization. Ifcrystallization is inhibited, so that more compound remains in solution,improved delivery may be achieved. Thus, the compositions providedherein may achieve rapid and sufficiently long-lasting solubilization oflow solubility benzoquinone ansamycin compounds in the aqueous medium inthe digestive tract, by inhibiting crystallization of the compound.

In some embodiments, the compositions containing benzoquinone ansamycincompounds (other than 17-DMAG), when dosed at a dose of 15 mg/kg ofactive compound, are capable of delivering an amount of compoundsufficient to achieve an AUC of at least 100 ng·ml/hr, at least 500ng·ml/hr, at least 1,000 ng·ml/hr, at least 5,000 ng·ml/hr, at least10,000 ng·ml/hr, at least 15,000 ng·ml/hr, at least 25,000 ng·ml/hr, orat least 50,000 ng·ml/hr of the active compound.

In some of the foregoing embodiments, the compound is present insubstantially amorphous form.

In some embodiments, a pharmaceutical composition for oraladministration is provided, comprising a crystallization inhibitor and acompound of formula 1:

or a pharmaceutically acceptable salt thereof,wherein;R¹ is H, —OR⁸, —SR⁸—N(R⁸)(R⁹), —N(R⁸)C(O)R⁹, —N(R⁸)C(O)OR⁹,—N(R⁸)C(O)N(R⁸)(R⁹), —OC(O)R⁸, —OC(O)OR⁸, —OS(O)₂R⁸, —OS(O)₂OR⁸,—OP(O)₂OR⁸, CN or a carbonyl moiety;each of R² and R³ independently is H, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, heterocycloaklyl, aryl, aralkyl, heteroaryl,heteroaralkyl, —C(═O)CH₃ or —[(C(R¹⁰)₂)_(p)]—R¹¹; or R² and R³ takentogether with the nitrogen to which they are bonded represent a 3-8membered optionally substituted heterocyclic ring which contains 1-3heteroatoms selected from O, N, S, and P;p independently for each occurrence is 0, 1, 2, 3, 4, 5, or 6;R⁴ is H, alkyl, akenyl, or aralkyl;R⁵ and R⁶ are each H; or R⁵ and R⁶ taken together form a bond;R⁷ is hydrogen alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,heterocycloaklyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or—[(C(R¹⁰)₂)_(p)]—R¹¹;each of R⁸ and R⁹ independently for each occurrence is H, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloaklyl, aryl,aralkyl, heteroaryl, heteroaralkyl, or —[(C(R¹⁰)₂)_(p)]—R¹¹; or R⁵ andR⁹ taken together represent a 3-8 membered optionally substitutedheterocyclic ring which contains 1-3 heteroatoms selected from O, N, S,and P;R¹⁰ for each occurrence independently is H, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, heterocycloaklyl, aryl, aralkyl, heteroaryl,or heteroaralkyl; andR¹¹ for each occurrence independently is H, cycloalkyl, aryl,heteroaryl, heterocyclyl, —OR⁸, —SR⁸, —N(R⁸)(R⁹), —N(R⁸)C(O)R⁹,—N(R⁸)C(O)OR⁹, —N(R⁸)C(O)N(R⁸)(R⁹), —OC(O)R⁸, —OC(O)OR⁸, —OS(O)₂R⁸,—OS(O)₂OR⁸, —OP(O)₂OR⁸, —C(O)R⁸, —C(O)₂R⁸, —C(O)N(R⁸)(R⁹), halide, orCN.

In some embodiments R¹ is OH, R⁴ is H, and R⁵ and R⁶ taken together forma bond.

In some embodiments, a pharmaceutical composition for oraladministration is provided, comprising a crystallization inhibitor and acompound of formula 1:

In certain embodiments, a pharmaceutical composition for oraladministration is provided, comprising a crystallization inhibitor and acompound of formula 1:

or a pharmaceutically acceptable salt thereof;wherein;R¹ is —OR⁸, —C(═O)CH₃, or a carbonyl moiety;each of R² and R³ independently is H, alkyl, alkenyl or—[(C(R¹⁰)₂)_(p)]—R¹¹; or R² and R³ taken together with the nitrogen towhich they are bonded represent a 3-8 membered optionally substitutedheterocyclic ring which contains 1-3 heteroatoms selected from O, N, S,and P;p independently for each occurrence is 0, 1 or 2;R⁴ is H;R⁵ and R⁶ are each H; or R⁵ and R⁶ taken together form a bond;R⁷ is hydrogen or —[(C(R¹⁰)₂)_(p)]—R¹¹;each of R⁸ and R⁹ independently are H; or R⁸ and R⁹ taken togetherrepresent a 3-8 membered optionally substituted heterocyclic ring whichcontains 1-3 heteroatoms selected from O, N, S, and P;R¹⁰ for each occurrence independently is H; andR¹¹ for each occurrence independently is H, —N(R⁸)(R⁹) or halide.

Examples of benzoquinone ansamycin compounds include those having thefollowing structures:

In some embodiments, compositions provided herein containing amorphous17-AG resulted in a surprising finding of improved bioavailabilityrelative to crystalline 17-AG even when no crystallization inhibitor wasused; such compositions are therefore useful for administration, such asoral administration.

In some of the foregoing embodiments, the compound is present insubstantially amorphous form.

Similarly, in some embodiments, the composition contains an amount ofcrystallization inhibitor of at least about 10%, 25%, 50%, 75% (w/w),based on the total weight of the composition.

In some of the foregoing embodiments, the crystallization inhibitor isPVP. In some of the foregoing embodiments, the 17-AG is substantiallyamorphous.

In certain embodiments, the pharmaceutical composition may be in theform of a paste, solution, slurry, ointment, emulsion or dispersion. Incertain embodiments, the pharmaceutical composition is, or comprises, amolecular dispersion.

In certain embodiments, the crystallization inhibitor may be selectedfrom polyvinylpyrrolidone (PVP) (including homo- and copolymers ofpolyvinylpyrrolidone and homopolymers or copolymers ofN-vinylpyrrolidone); crospovidone; gums; cellulose derivatives(including hydroxypropyl methylcellulose (HPMC), hydroxypropylmethylcellulose phthalate, hydroxypropyl cellulose, ethyl cellulose,hydroxyethylcellulose, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, sodium carboxymethyl cellulose, and others);dextran; acacia; homo- and copolymers of vinyllactam, and mixturesthereof; cyclodextrins; gelatins; hypromellose phthalate; sugars;polyhydric alcohols; polyethylene glycol (PEG); polyethylene oxides;polyoxyethylene derivatives; polyvinyl alcohol; propylene glycolderivatives and the like, SLS, Tween, EUDRAGIT (a methacrylic acid andmethyl methacrylate copolymer); and combinations thereof. Thecrystallization inhibitor may be water soluble or water insoluble.

HPMCs vary in the chain length of their cellulosic backbone andconsequently in their viscosity as measured for example at a 2% (W/W) inwater. HPMC used in the pharmaceutical compositions provided herein mayhave a viscosity in water (at a concentration of 2% (w/w)), of about 100to about 100,000 cP, about 1000 to about 15,000 cP, for example about4000 cP. In certain embodiments, the molecular weight of HPMC used inthe pharmaceutical compositions provided herein may have greater thanabout 10,000, but not greater than about 1,500,000, not greater thanabout 1,000,000, not greater than about 500,000, or not greater thanabout 150,000.

HPMCs also vary in the relative degree of substitution of availablehydroxyl groups on the cellulosic backbone by methoxy and hydroxypropoxygroups. With increasing hydroxypropoxy substitution, the resulting HPMCbecomes more hydrophilic in nature. In certain embodiments, the HPMC hasabout 15% to about 35%, about 19% to about 32%, or about 22% to about30%, methoxy substitution, and having about 3% to about 15%, about 4% toabout 12%, or about 7% to about 12%, hydroxypropoxy substitution.

HPMCs which can be used in the pharmaceutical compositions areillustratively available under the brand names Methocel™ of Dow ChemicalCo. and Metolose™ of Shin-Etsu Chemical Co. Examples of suitable HPMCshaving medium viscosity include Methocel™ E4M, and Methocel™ K4M, bothof which have a viscosity of about 400 cP at 2% (w/w) water. Examples ofHPMCs having higher viscosity include Methocel™ E10M, Methocel™ K15M,and Methocel™ K100M, which have viscosities of about 10,000 cP, 15,000cP, and 100,000 cP respectively viscosities at 2% (w/w) in water. Anexample of an HPMC is HPMC-acetate succinate, i.e., HPMC-AS.

In certain embodiments the PVPs used in pharmaceutical compositionsprovided herein have a molecular weight of about 2,500 to about3,000,000 Daltons, about 8,000 to about 1,000,000 Daltons, about 10,000to about 400,000 Daltons, about 10,000 to about 300,000 Daltons, about10,000 to about 200,000 Daltons, about 10,000 to about 100,000 Daltons,about 10,000 to about 80,000 Daltons, about 10,000 to about 70,000Daltons, about 10,000 to about 60,000 Daltons, about 10,000 to about50,000 Daltons, or about 20,000 to about 50,000 Daltons. In certaininstances the PVPs used in pharmaceutical compositions provided hereinhave a dynamic viscosity, 10% in water at 20° C., of about 1.3 to about700, about 1.5 to about 300, or about 3.5 to about 8.5 mPas.

When PEGs are used they can have an average molecular about 5,000-20,000Dalton, about 5,000-15,000 Dalton, or about 5,000-10,000 Dalton.

Also provided herein is a pharmaceutical composition for oral delivery,comprising 17-AG and at least one pharmaceutically acceptable excipient,wherein said pharmaceutical composition is substantially free ofcrystalline 17-AG. In certain instances, the 17-AG in such apharmaceutical composition includes less than about 15% (w/w), less thanabout 10% (w/w), less than about 5% (w/w), less than about 3% (w/w), orless than about 1% (w/w) crystalline 17-AG. Such a pharmaceuticalcomposition may be formulated as a solid dosage form (e.g., a tablet orcapsule), a paste, emulsion, slurry, or ointment.

Also provided herein is a pharmaceutical composition for oral delivery,comprising 17-AAG and at least one pharmaceutically acceptableexcipient, wherein said pharmaceutical composition is substantially freeof crystalline 17-AAG. In certain instances, the 17-AAG in such apharmaceutical composition includes less than about 15% (w/w), less thanabout 10% (w/w), less than about 5% (w/w), less than about 3% (w/w), orless than about 1% (w/w) crystalline 17-AAG. Such a pharmaceuticalcomposition may be formulated as a solid dosage form (e.g., a tablet orcapsule), a paste, emulsion, slurry, or ointment.

As described above, benzoquinone ansamycins and pharmaceuticalcompositions of the present invention may additionally comprisepharmaceutically acceptable carriers and excipients according toconventional pharmaceutical compounding techniques to form apharmaceutical composition or dosage form. Suitable pharmaceuticallyacceptable carriers and excipients include, but are not limited to,those described in Remington's, The Science and Practice of Pharmacy,(Gennaro, A. R., ed., 19th edition, 1995, Mack Pub. Co.), which isherein incorporated by reference. The phrase “pharmaceuticallyacceptable” refers to additives or compositions that are physiologicallytolerable and do not typically produce an allergic or similar untowardreaction, such as gastric upset, dizziness and the like, whenadministered to an animal, such as a mammal (e.g., a human). For oralliquid pharmaceutical compositions, pharmaceutical carriers andexcipients can include, but are not limited to water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents, and thelike. Oral solid pharmaceutical compositions may include, but are notlimited to starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders and disintegrating agents. Thepharmaceutical composition and dosage form may also include abenzoquinone ansaymyscin compound or solid form thereof as discussedabove.

The solid forms described herein can be useful for making pharmaceuticalcompositions suitable for oral administration. Such pharmaceuticalcompositions may contain any of the benzoquinone ansamycin compoundsdescribed herein, for example, in an amorphous form and nocrystallization inhibitor, or an amorphous form in combination with acrystallization inhibitor. Examples of such benzoquinone ansamycins aredescribed in Schnur et al., J. Med. Chem. 1995, 38: 3806-12.

(4) Pharmaceutical Uses and Methods of Treatment

Also provided herein are methods of treating cancer, inhibiting Hsp90,and/or treating a hyperproliferative disorder comprising orallyadministering to a patient in need thereof a therapeutically effectiveamount of any of the aforementioned compounds or pharmaceuticalcompositions. For example, 17-AAG is currently being studied in clinicaltrials as a treatment for multiple myeloma. 17-AG is produced in thehuman body by metabolism of 17-AAG (Egorin et at 1998) and is alsobelieved to be an active anti-cancer agent. The cancer, neoplasticdisease state or hyperproliferative disorder is selected from the groupconsisting of gastrointestinal stromal tumor (GIST), colon cancer,colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, small cell lung cancer, non-small cell lung cancer,melanoma, multiple myeloma, myelodysplastic syndrome, acute lymphocyticleukemia, acute myelocytic leukemia, chronic myelocytic leukemia,chronic lymphocytic leukemia, polycythemia Vera, Hodgkin lymphoma,non-Hodgkin lymphoma, Waldenstrom's macroglobulinemia, heavy chaindisease, soft-tissue sarcomas, such as fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, stadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,neuroblastoma, retinoblastoma, endometrial cancer, follicular lymphoma,diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellularcarcinoma, thyroid cancer, gastric cancer, esophageal cancer, head andneck cancer, small cell cancers, essential thrombocythemia, agnogenicmyeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis,familiar hypereosinophilia, chronic eosinophilic leukemia, thyroidcancer, neuroendocrine cancers, and carcinoid tumors.

In certain embodiments, the cancer is selected from the group consistingof gastrointestinal stromal tumor, multiple myeloma, prostate cancer,breast cancer, melanoma, chronic myelocytic leukemia, and non-small celllung cancer.

In certain embodiments, the methods described herein treat a diseaseusing a benzoquinone compound such as 17-AG. In certain embodiments,17-AG is substantially amorphous.

(5) Dosing

Actual dosage levels of the benzoquinone ansamycins, e.g., geldanamycinanalogs, in the pharmaceutical compositions of the present invention maybe varied so as to obtain an amount of the gelanamycin analog which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular geldanamycin analog employed,or salt thereof, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the rate and extent of absorption, the durationof the treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds provided herein, employed in thepharmaceutical composition, at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable dose of a geldanamycin analog will be that amountof the compound which is the lowest safe and effective dose to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. When the geldanamycin analogs areadministered in combination with another chemotherapeutic or withradiation, the doses of each agent will in most instances be lower thanthe corresponding dose for single-agent therapy.

Provided compositions may be formulated into a unit dosage form. Suchformulations are well known to one of ordinary skill in the art andinclude capsules, tablets, and the like. In certain embodiments, thepresent invention provides a formulation comprising a capsule filledwith inventive geldanamycin analogs. In other embodiments, the presentinvention provides a capsule for oral administration comprisinginventive geldanamycin analogs. In some embodiments, a unit dosage form(e.g., a capsule or tablet) contains 5-1,000 mg, e.g., 25, 50, 125, 250or 500 mg, of a geldanamycin analog. In some embodiments, a unit dosageform contains more than 5 mg/kg of a geldanamycin analog.

In some embodiments, the oral dose is between 1 mg/kg and 100 mg/kg,inclusive, or between 5 mg/kg and 50 mg/kg, inclusive, or between 5mg/kg and 25 mg/kg, inclusive, or between 10 mg/kg and 20 mg/kg,inclusive, of a geldanamycin analog characterized in that the area underthe curve of at least 100 ng·hr/ml is achieved. In some embodiments, thedose is 15 mg/kg. In some embodiments, the area under the curve achievedis at least 500, 1000, 5000, 10,000, or 15,000 ng·hr/ml.

A total daily dosage of a geldanamycin analog (e.g., 17-AG or 17-AAG)will typically be in the range 500-1,500 mg per day. In certainembodiments, an effective amount of a geldanamycin analog foradministration to a 70 kg adult human may comprise about 100 mg to about1,500 mg of compound (e.g., 17-AG or 17-AAG) per day. It will beappreciated that dose ranges set out above provide guidance for theadministration of active compound to an adult. The amount to beadministered to, for example, an infant or a baby can be determined by amedical practitioner or person skilled in the art and can be lower orthe same as that administered to an adult.

The geldanamycin analog can be administered daily, every other day,three times a week, twice a week, weekly, or bi-weekly. The dosingschedule can include a “drug holiday,” i.e., the drug can beadministered for two weeks on, one week off, or continuously, without adrug holiday.

(6) Combination Therapy

In some embodiments, the pharmaceutical compositions described hereincan be used in combination with other therapeutic agents in order toachieve selective activity in the treatment of cancer. In certainembodiments, the geldanamycin analogs described herein are used toreduce the cellular levels of properly folded Hsp90 client proteins,which are then effectively inhibited by the second agent. For example,binding of a benzoquinone ansamycin analog to Hsp90 results in targetingof the client protein to the proteasome, and subsequent degradation.Using an agent that targets and inhibits the proteasome, e.g., Velcade™,then leads to increased cellular apoptosis and cell death.

Some examples of therapeutic agents which can be used in combinationwith the formulations described herein include alkylating agents;anti-angiogenic agents; anti-metabolites; epidophyllotoxin;procarbazine; mitoxantrone; platinum coordination complexes;anti-mitotics; biological response modifiers and growth inhibitors;hormonal/anti-hormonal therapeutic agents; haematopoietic growthfactors; the anthracycline family of drugs; the vinca drugs; themitomycins; the bleomycins; the cytotoxic nucleosides; the epothilones;discodermolide; the pteridine family of drugs; diynenes; and thepodophyllotoxins. Particularly useful members of those classes include,for example, caminomycin, daunorubicin, aminopterin, methotrexate,methopterin, dichloromethotrexate, mitomycin C, porfiromycin,5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside,podophyllotoxin or podophyllotoxin derivatives such as etoposide,etoposide phosphate or teniposide, melphalan, vinblastine, vincristine,leurosidine, doxorubicin, vindesine, leurosine, paclitaxel, taxol,taxotere, docetaxel, cis-platin, imatinib mesylate, or gemcitebine.

Other useful agents include estramustine, carboplatin, cyclophosphamide,bleomycin, gemcitibine, ifosamide, melphalan, hexamethyl melamine,thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine,L-asparaginase, camptothecin, CPT-11, topotecan, ara-C, bicalutamide,flutamide, leuprolide, pyridobenzoindole derivatives, interferons andinterleukins. Particularly useful agents include taxotere, Gleevec(imatinib), Tarceva (erlotinib), Sutent (sunitinib), Tykerb (lapatinib),and Xeloda (capecitabine).

The formulations described herein can also be used in conjunction withradiation therapy. The chemotherapeutic agent/radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent and/or radiation therapy on that disease. Thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents (i.e., antineoplastic agent or radiation) on thepatient, and in view of the observed responses of the disease to theadministered therapeutic agents.

Also, in general, the geldanamycin analogs described herein and thesecond chemotherapeutic agent do not have to be administered in the samepharmaceutical composition, and may, because of different physical andchemical characteristics, have to be administered by different routes.For example, the geldanamycin compound can be administered orally, whilethe second chemotherapeutic is administered intravenously. Thedetermination of the mode of administration and the advisability ofadministration, where possible, in the same pharmaceutical composition,is well within the knowledge of the skilled clinician. The initialadministration can be made according to established protocols known inthe art, and then, based upon the observed effects, the dosage, modes ofadministration and times of administration can be modified by theskilled clinician.

The particular choice of chemotherapeutic agent or radiation will dependupon the diagnosis of the attending physicians and their judgment of thecondition of the patient and the appropriate treatment protocol.

The geldanamycin analog and the second chemotherapeutic agent and/orradiation may be administered concurrently (e.g., simultaneously,essentially simultaneously or within the same treatment protocol) orsequentially, depending upon the nature of the proliferative disease,the condition of the patient, and the actual choice of chemotherapeuticagent and/or radiation to be administered in conjunction (i.e., within asingle treatment protocol) with the geldanamycin analog.

If the geldanamycin analog, and the chemotherapeutic agent and/orradiation are not administered simultaneously or essentiallysimultaneously, then the optimum order of administration may bedifferent for different tumors. Thus, in certain situations thegeldanamycin analog may be administered first followed by theadministration of the chemotherapeutic agent and/or radiation; and inother situations the chemotherapeutic agent and/or radiation may beadministered first followed by the administration of a geldanamycinanalog. This alternate administration may be repeated during a singletreatment protocol. The determination of the order of administration,and the number of repetitions of administration of each therapeuticagent during a treatment protocol, is well within the knowledge of theskilled physician after evaluation of the disease being treated and thecondition of the patient. For example, the chemotherapeutic agent and/orradiation may be administered first, especially if it is a cytotoxicagent, and then the treatment continued with the administration of ageldanamycin analog followed, where determined advantageous, by theadministration of the chemotherapeutic agent and/or radiation, and so onuntil the treatment protocol is complete.

Thus, in accordance with experience and knowledge, the practicingphysician can modify each protocol for the administration of a component(therapeutic agent, i.e., geldanamycin analog, chemotherapeutic agent orradiation) of the treatment according to the individual patient's needs,as the treatment proceeds.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular geldanamycin analog employed,or salt thereof, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the rate and extent of absorption, the durationof the treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds provided herein, employed in thepharmaceutical composition, at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable dose of a geldanamycin analog will be that amountof the compound which is the lowest safe and effective dose to produce atherapeutic effect. The dose can be 1 mg/kg to 25 mg/kg. Such aneffective dose will generally depend upon the factors described above.When the geldanamycin analogs are administered in combination withanother chemotherapeutic or with radiation, the doses of each agent willin most instances be lower than the corresponding dose for single-agenttherapy.

EXAMPLES

The present disclosure now being generally described, it will be morereadily understood by reference to the following examples, which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit thedisclosure herein.

In general, geldanamycin analogs are known to be Hsp90 inhibitors(Schnur et al., J. Med. Chem. (1995), Vol. 38, pages 3806-3812).Examples 1 through 11 describe synthetic chemical preparation forvarious geldanamycin analogs and solid forms thereof.

Example 1 Preparation of Form I of 17-AG

A 22 L RB flask was equipped with a bottom drain valve, mechanicalstirring, a 1 L addition funnel, internal temperature probe, and aninert gas bypass. Geldanamycin (500 g, 1 eq) and anhydrous THF (5.0 L)was charged to a 22 L RB flask. The stirring is started and Ammonia inMeOH (7 M) is charged (1.0 L, 8.0 eq.) The reaction was stirred atambient temperature 7 hours. The LCMS indicated complete consumption ofstarting material at 7 hours. During the course of the reaction, thecolor changed from yellow to deep purple. Heptane (14 L) was slowlyadded to the reaction mixture, inducing crystallization of the desiredproduct from solution. The brick red slurry was stirred overnight. Theproduct was isolated by suction filtration and rinsed wth 2:1 (v/v)heptane/THF (0.5 L). Oven drying provided crude 17-AG as a powdery,dingy red solid (470 g). The crude material is dissolved in a 4:1mixture of acetone/ethanol (18-19 L) with heating and clarified. Thesolution is concentrated and solvent exchanged with additional ethanol(2 L). The ethanol slurry of purple solids is diluted with ethanol (4 L)and water (5 L). The slurry is aged overnight at 35° C. and then heatedto 70° C. for 3 hours, during which the crystal form changes and thecolor turns from dark purple to red. The slurry is cooled to roomtemperature and the solids are isolated by filtration. The Karl Fisheranalysis was 0.86% and all residual solvents were low (EtOH 2266 ppm;acetone 89 ppm; heptane 9 ppm; THF and MeOH not detected). This is thepolymorph referred to as Form I.

Example 2 Preparation of Form II of 17-AG

Form I 17-AG (10 g) from the preceding procedure was dissolved inacetone/ethanol at 30° C. and clarified. The flask and the in-linefilter were rinsed, and the solution was concentrated via a rotovap to athick slurry. Then 100 mL of water was added and the rest of the organicsolvents removed by vacuum distillation. When the distillate collectionceased, the bath temperature was increased from 40° C. to 60° C. and asmall amount of water was removed. Then another portion of water wasadded (100 mL). With a bath temperature of 80° C. and slight vacuum,water was distilled for ca. 5 min. The slurry remained purple, so thevacuum was disconnected, and the bath increased to 100° C. The slurrywas mixed for ca 1 h. The slurry was then allowed to cool to ambienttemperature overnight and the purple solids were isolated from water.The Karl Fisher analysis was 0.14% and all residual solvents were low(MeOH: 106 ppm, EtOH: 173 ppm, Acetone 230 ppm, and THF and heptane werenot detected). This material is the Form TI polymorph.

Example 3 Preparation of Form III of 17-AG

To 400 mL of distilled water was added 1 g of 20% solid dispersion of17-aminogeldanamycin in PVP K-30 (as prepared in Example 14). Thesuspension was heated to 60° C. until complete dissolution of the solid.After heating at 60° C. for 5-10 min, purple crystals precipitated fromthe solution. The mixture was allowed to cool to 23° C. and the purplecrystalline material was isolated by filtration. The collected crystalswere dried for 2 days in vacuum oven at 80° C. to give 155 mg 17-AG FormIII as purple powder. Yield 75%. MS (ESI(+)) m/z 563.4 (M+H₂O)⁺.

Example 4 Preparation of 17-AG Ethyl Acetate Solvate

To 17-AG (1.2 g) (Form I Polymorph) was added EtOAc (150 mL). Themixture was heated a gentle reflux until 17-AG completely dissolved. Thesolutions were analyzed using polarized light microscopy to ensurecomplete dissolution. The volume was reduced to ca 5 mL using a rotatoryevaporator and the solution was allowed to cool slowly to roomtemperature. After 12 h, the mixture was filtered, washed with hexanesand dried to provide the EtOAc solvate based on ¹HNMR.

Example 5 Preparation of 11-oxo-17-aminogeldanamycin

To a 23° C. solution of 17-aminogeldanamycin (5.0 g, 9.16 mmol, 1.0 eq)in CHCl₃ (750 mL) was added Dess-Martin periodinane (23.32 g, 55.0 mmol,6.0 eq.) in a single portion. After stirring for 30 min, the reactionmixture was diluted with CHCl₃, washed with aqueous sodium thiosulfateand saturated aqueous sodium bicarbonate. The organic layer wasseparated, dried over sodium sulfate, filtered and concentrated invacuo. The crude material was further purified by recrystallization(DCM/Hexane) to afford 4.12 g of the pure desired product. Yield 83%. MS(ESI(+)) m/z 566.3 (M+Na)⁺.

Example 6 Preparation of 11-acetyl-17-aminogeldanamycin

To a 23° C. solution of 17-aminogeldanamycin (6.0 g, 11.0 mmol, 1.0 eq)in anhydrous DCM (156 mL) under nitrogen atmosphere was added aceticanhydride (2.075 mL, 21.99 mmol, 2.0 eq.), DMAP (1.343 g, 11.0 mmol, 1.0eq.) and triethylamine (4.60 mL, 33.0 mmol, 3.0 eq.). The reactionmixture was allowed to stir overnight. The reaction mixture was dilutedwith DCM (200 mL), washed with water (100 mL) and brine (2×100 mL),dried with Na₂SO₄, filtered and concentrated in vacuo. The crudematerial was purified by gradient flash chromatography (SiO₂, 30%-60%EtOAc/Hexanes) to provide 1.9 g of the desired product with a traceamount of tris-acetylated product. Yield 30.9%. MS (ESI(+)) m/z 610.4(M+Na)⁺.

Example 7 Preparation of 17-cyclopropylmethylaminogeldanamycin

To a 23° C. solution of geldanamycin (3.0 g, 5.35 mmol, 1.0 eq) in DCM(54 mL) under argon was added cyclopropanemethylamine (9.40 mL, 107mmol, 20 eq). The reaction mixture was allowed to stir for 2 hours. Thereaction mixture was then quenched with water (100 mL) and acidifiedwith 1 N HCl to pH 3 and stirred for an additional 30 minutes. Theorganic layer was separated and the aqueous layer was extracted withDCM. The combined organic extracts were washed with water, dried overNa₂SO₄, filtered and concentrated in vacuo. The crude product waspurified using gradient flash chromatography (SiO₂, 50-60%EtOAc/Hexanes) to afford 2.7 g of the desired product. Yield 84.0%. MS(ESI(+)) m/z 622.4 (M+Na)⁺.

Example 8 Preparation of 17-benzylaminogeldanamycin

To a 23° C. solution of geldanamycin (3.25 g, 5.35 mmol, 1.0 eq) in DCM(110 mL) under argon was added benzylamine (9.40 mL, 53.5 mmol, 10 eq)in a single portion. After stirring at 23° C. for 12 h, the reactionmixture was diluted with water (100 mL) and acidified with 1 N HCl to pH3 and stirred for an additional 30 minutes. The organic layer wasseparated and the aqueous layer was extracted with DCM. The combinedorganic extracts were washed with water, dried over Na₂SO₄, filtered andconcentrated in vacuo. The crude product was purified using gradientflash chromatography (SiO₂, 50-60% EtOAc/Hexanes) to afford 3.51 g ofthe product. Yield 95.0%. MS (ESI(+)) m/z 658.4 (M+Na)⁺.

Example 9 Preparation of 17-azetidinylgeldanamycin

To a 23° C. solution of azetidine hydrochloride (751 mg, 8.03 mmol, 2.0eq.) in 1:1 DCM:Methanol (100 mL) was added Hunig's base (2.10 mL, 12.04mmol, 3.0 eq) followed by geldanamycin (2.25 g, 4.01 mmol, 1.0 eq).After stirring at 23° C. for 2 hours, the reaction mixture wasconcentrated in vacuo and then re-dissolved in DCM (100 mL). Water (100mL) was added and the aqueous layer was acidified to pH 3. The mixturewas then stirred for 30 minutes. The organic layer was separated and theaqueous layer was extracted with DCM (3×100 mL). The combined organiclayers were washed with water (300 mL), dried over MgSO₄, filtered andconcentrated in vacuo. The crude material was further purified viarecrystallization from Chloroform/Hexane to afford 1.92 g of the puredesired product. Yield 82%. MS (ESI(+)) m/z 586.1 (M+H)⁺.

Example 10 Preparation of 17-(fluoroethyl)aminogeldanamycin

To a 23UC solution of 2-fluoroethylamine hydrochloride (7.99 g, 80.25mmol, 7.5 eq.) in DCM (240 mL) and MeOH (120 mL) under nitrogenatmosphere was added Hunig's base (14.02 mL, 80.25 mmol, 7.5 eq.). Afterthe 2-fluoroethylamine hydrochloride dissolved, geldanamycin (6.0 g,10.70 mmol, 1.0 eq)) was added. After stirring for 24 h at 23° C., thereaction mixture was concentrated in vacuo and then re-dissolved in DCM(900 mL). Water (300 mL) was added and the aqueous layer was acidifiedto pH 3. The mixture was stirred for 30 minutes. The organic layer wasseparated and the aqueous layer was extracted with DCM (3×100 mL). Thecombined organic layers were washed with water (900 mL), dried overNa₂SO₄, filtered and concentrated in vacuo. The crude product waspurified using gradient flash chromatography (SiO₂, 30-60% EtOAc/DCM) toafford 3.0 g of the desired product. Yield 47.4%. MS (ESI(+)) m/z 614.4(M+Na)⁺.

Example 11 Preparation of 17-acetylgeldanamycin

To a 23° C. solution of 17-aminogeldanamycin (5.5 g, 10.08 mmol, 1.0 eq)in EtOAc (500 mL) was added Na₂S₂O₄ (0.1 M, 500 mL). The biphasicmixture was stirred at 23° C. until the reaction mixture went from adeep purple to a pale yellow color (ca 10 min). The organic layer wasseparated and the aqueous layer was extracted with EtOAc (3×200 mL). Thecombined organic extracts were dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was then dissolved in CHCl₃ (72 mL)under an inert atomosphere (N₂) and cooled to 0° C. using an ice bath.Acetic anhydride (2.85 mL, 30.2 mmol, 3.0 eq.) was added dropwise at 0°C. After stirring for 3 h, the reaction mixture was diluted with EtOAcand concentrated in vacuo. The crude product was dissolved in methanolat 23° C. and stirred for 4 days under an open atmosphere to allow forthe oxidation of the hydroquinone to the quinone. The crude product waspurified by isocratic flash chromatography (80:15:5 DCM:EtOAc:MeOH) toafford 3.5 g of the desired product as a yellow solid. Yield 59.1%. MS(ESI(+)) m/z 610.4 (M+Na)⁺.

Example 12

Oral Bioavailabilty Effects upon Administration of Amorphous DispersionFormulation (17-AG plus PVP)

The effect on oral bioavailability of an exemplary compound, 17-AG, inthe form of an amorphous dispersion of 17-AG plus PVP(polyvinylpyrrolidone, or also referred to as Povidone) was investigatedby dosing beagle dogs and measuring 17-AG levels in blood plasma atvarious time points following a single oral capsule dose.

A 12% 17-AG/PVP (w/w) dispersion was made utilizing rotary evaporationand characterized for purity, residual solvent level, and amorphouscontent as described, filled into HPMC capsules and dosed into dogs at alevel of 15 mg/kg. Blood was collected pre-dose, at 15 minutes, at 30minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours postdose into tubes containing sodium heparin. Collected blood samples wereimmediately placed on wet ice and refrigerated centrifuge for isolationof plasma within 30 min of collection. Isolated plasma was saved inlabeled screw cap freezer vials or eppendorf tubes and stored frozen(−70° C.) until analyzed for plasma 17-AG levels. The design of thestudy is as follows. A single group of dogs consisting of 2 males & 2females was utilized for dosing, with a week washout in between eachdose.

Dose 1 Dose 2 Dose 3 Dose 4 12% 17-AG PVP N/A Crystalline 17-AG 12%17-AG PVP K30 @15 mg/kg, @15 mg/kg, uncoated K30 @15 mg/kg, uncoatedHPMC HPMC capsule enteric coated capsule HPMC capsule

After analysis of 17-AG plasma levels following dosing, there was asignificant effect on exposure due to the dosing of amorphous 17-AG/PVPdispersion. There was >100-fold increase in both Cmax and AUC levelswhen dosing amorphous 17-AG/PVP dispersion as compared to dosingcrystalline 17-AG. Plasma levels of 17-AG following dosing ofcrystalline material were below quantifiable limits. Dosing of the17-AG/PVP dispersion in coated capsules also produced similar increasesin exposure but was not as significant as the result from the uncoatedcapsule. There were no differences seen in the exposure data due to sexfor the variables tested. Any variability observed in the exposure ismost likely due to animal specific differences or in-life observations(i.e. dosing issues, emesis).

Oral bioavailability results (average values) are found in FIG. 1 a(male) and FIG. 11 a (female), which demonstrate that crystalline 17-AGhas very low oral bioavailability, but amorphous compound has highbioavailability. Summary Tables of PK data are shown in FIG. 10 b andFIG. 11 b, respectively.

Following are exemplary methods of preparing solid dispersionformulations using amorphous geldanamycin analogs. Generally, eachformulation may be prepared either with a crystallization inhibitor, orwithout a crystallization inhibitor. When present, the crystallizationinhibitor used may vary in both type and in amount. Exemplary methodsinclude, but are not limited to cryo-grinding (Example 13(a)), spraydrying (Example 13(b)), lyophilization (Example 13(c)) and rotaryevaporation (Example 14 through Example 16). An exemplary DSC patternthat resulted from one technique, i.e., lyophilization utilizing t-BuOH,is found in FIG. 12. Exemplary Exposure data using two different methodsof preparing solid dispersions are found in FIG. 14 a (rotaryevaporation) and FIG. 14 b (spray-dried) and a Summary Table in FIG. 14c.

Example 13 Preparation of Amorphous 17-AG

To 17-AG (1 g) (Form I Polymorph) was added CH₃CN (50 mL) followed byt-BuOH (100 mL). The mixture was heated at 60° C. until 17-AG completelydissolved was then clarified by filtration through a 0.45 um filter. Thesolutions were analyzed using polarized light microscopy to ensurecomplete dissolution. The filtrate was immersed into a bath of liquidnitrogen until frozen and then lyophilized, which resulted in amorphous17-AG as a light purple powder. The amorphous nature was confirmed viapolarized light microscopy.

Example 13(a) Cryo-Grinding Protocol Preparation of Amorphous SolidDispersion Formulation (17-AG and PVP)

17-AG (˜1 g) was cooled to ˜200° C. in liquid nitrogen and ground toproduce amorphous material by physical grinding and pulverizing for 30minutes. Ground samples were checked for amorphous state by XRPD andP.L.M.

Example 13(b) Spray Drying Protocol Preparation of Amorphous SolidDispersion Formulation [17-AG (20% load) plus Polyvinylpyrrolidone (PVP)K-30 Povidone®]

To a 3:1 mixture of acetone (75 g, 94.95 mL) and ethanol 190 proofUSP/NF grade (25 g, 31.65 mL) was added Polyvinylpyrrolidone (PVP) K-30Povidone® (20 g) in a single portion. The mixture was stirred at 23° C.until the dissolution of the polymer was complete (ca 30 min). 17-AG (5g, 36.7 mmol) was added in portions over the course of 10 mins toprovide an opaque purple mixture. After stirring for 2 hours at the roomtemperature, an aliquot was examined using PLM to ensure completedissolution; the purple solution was then spray-dried on a Buchi minispray dryer under the following conditions; Inlet temperature 90° C.,Outlet temperature 64° C., N₂ flow 600 l/h, Aspiration 70%, to provide alight purple amorphous powder. This material was amorphous based onanalysis via polarized light microscopy. MS (ESI(+)) m/z 563.4 (M+H₂O)⁺.

Example 13(c) Lyophilization Protocol Preparation of Amorphous SolidDispersion Formulation [17-AG in PVP]

A series of amorphous dispersions of varying loads of 17-AG (12%, 15%,20%, 30%, 40%, 50% w/w) in Polyvinylpyrrolidone (PVP) K-30 and K-90Povidone® were prepared via lyophilization according to the followingprotocol. A representative DSC pattern is found in FIG. 12.

A mixture of 17-AG (100 mg, 0.18 mmol) in t-BuOH (500 mL) was heated to50° C. using a heat gun and stirred vigorously. Aliquots wereperiodically taken and examined via polarized light microscopy todetermine complete dissolution of crystalline 17-AG. After heating at50° C. for 2 h, the dissolution was complete due to a lack ofbirefringence observed under polarized light microscopy. The 17-AGsolution (62.5 mL, 12.5 mg) was slowly added to the appropriate amountof PVP (K-30 or K-90) dissolved in water (20.8 mL) according to thefollowing schedule. All mixtures maintained a ratio of t-BuOH:water(3:1).

-   -   12% load-17-AG (12.5 mg) in tBuOH (62.5 mL) PVP (91.5 mg) in        water (20.8 mL)    -   15% load-17-AG (12.5 mg) in tBuOH (62.5 mL) PVP (71 mg) in water        (20.8 mL)    -   20% load-17-AG (12.5 mg) in tBuOH (62.5 mL) PVP (50 mg) in water        (20.8 mL)    -   30% load-17-AG (12.5 mg) in tBuOH (62.5 mL) PVP (29 mg) in water        (20.8 mL)    -   40% load-17-AG (12.5 mg) in tBuOH (62.5 mL) PVP (18.7 mg) in        water (20.8 mL)    -   50% load-17-AG (12.5 mg) in tBuOH (62.5 mL) PVP (12.5 mg) in        water (20.8 mL)        The warm solutions were then transferred to lyophilization cups        (110 mL capacity) and allowed to slowly cool to 23° C. After        reaching 23° C., the solutions were again analyzed using        polarized light microscopy to ensure complete dissolution. No        birefringence was detected. All cups were transferred to a        pre-cooled (−40° C.) tray lyophilizer, held at −40° C. for 8        hours, and then slowly ramped to 23° C. over the course of 2        days resulting in light purple amorphous solids in quantitative        yield. All samples were amorphous based on polarized light        microscopy, and >95% pure by HPLC.

Following Examples 14 through 16 further illustrate the rotaryevaporation technique to prepare solid dispersion formulations utilizinga variety of PVP grades.

Example 14 Rotary Evaporation Protocol Preparation of Amorphous SolidDispersion Formulation [17-AG (12%, 20% and 30% load) and PVP K-30]

17-AG (12% load, 1.52 g) was added to ethanol (200 proof, 100 mL) andthe mixture was stirred at 45° C. for 45 min. In a separate flask PVPK-30 (11.13 g) was added to ethanol (200 proof, 150 mL). The resultingsolution was stirred at 45° C. for 15 min. The 17-AG solution was addedto the PVP solution and the resulting solution was stirred at 45° C. foran additional 4 hours (monitored using a microscope, looking for totaldisappearance of crystals). The homogeneous purple solution was thenconcentrated and pumped under high vacuum for 12 hours. The resultingglass material was analyzed by ¹H-NMR (to determine residual ethanolcontent) and by Cross Polar Microscopy (to determine the amount ofresidual crystalline material). The material was crushed to a powderusing a mortar and a pestle and dried under high vacuum at 40° C. for 10h after which time it was analyzed by ¹H-NMR for ethanol content. Thematerial was crushed again and further dried under high vacuum for anadditional 16 hours to result in fine glassy red-purple material (11.1g) containing 3% w/w of ethanol. Material was analyzed by ¹H-NMR, CPM,HPLC and DSC. The amount of 17-AG was adjusted accordingly to achievecorresponding 20% load of 17-AG (w/w) and 30% load of 17-AG (w/w).Representative dissolution data for 12%, 20% and 30% loads are found inFIG. 39.

Example 15 Rotary Evaporation Protocol Preparation of Amorphous SolidDispersion Formulation [17-AG (12% load) in PVP K-15]

17-AG (10 g) was added to EtOH (1 L, 99.9%) in a 3 L one-necked flask.This mixture was turned at ca 100 rpm on a rototary evaporator at 60° C.and atmospheric pressure. Aliquots were periodically taken and examinedvia polarized light microscopy to determine complete dissolution of thecrystalline 17-AG. After turning at 60° C. for 2 h, the dissolution wascomplete due to a lack of birefringence observed under polarized lightmicroscopy. Polyvinylpyrrolidone (PVP) K-15 Povidone® (73 g) was addedin a single portion and the mixture returned to the rototary evaporatorand turned at ca 100 rpm and 60° C. bath temperature. After 1 h, thesolutions were again analyzed using polarized light microscopy to ensurecomplete dissolution. No birefringence was detected. Vacuum was appliedand the EtOH was removed over the course of 30 min resulting in purplefoam. The flask was transferred to the hi-vac and dried overnight. Thebrittle foam was scraped from the sides and the crude material waspumped under hi-vac for an additional 36 h. The material was furthercrushed with a spatula to facilitate removal from the flask to provide76 g (92% yield) of an amorphous dispersion based on polarized lightmicroscopy and XRPD. Representative dissolution data is found in FIG.38.

Example 16 Rotary Evaporation Protocol Preparation of Amorphous SolidDispersion Formulation [17-AG (12% load) in PVP K-90]

An amorphous solid dispersion formulation [17-AG (12% load) in PVP K-90was prepared using a procedure similar to above Example 15 except thatPVP K-30 was used to obtain 48 g (48% yield) of an amorphous dispersionbased upon polarized light microscopy, using rotary evaporation followedby hi-vac. Representative dissolution data is found in FIG. 38.

Following Example 17 is a stability study that was conducted on theformulations described above (in Examples 14, 15 and 16), and summarizedin the table below.

Example 17

Various 17-AG/PVP dispersions were subjected to various storageconditions and the chemical and physical stability of each were assessedat specified time points. The dispersions tested were: 12% 17-AG in PVPK15, 12% 17-AG in PVP K90, 12% 17-AG in PVP K30, 20% 17-AG in PVP K30,and 30% 17-AG in PVP K30. The storage conditions tested were: RT ambienthumidity, RT 33% RH, RT 75% RH, 40° C. ambient humidity, and 40° C. 75%RH. Chemical stability of 17-AG was assessed by measurement of purity byRP-HPLC, and physical stability of the amorphous dispersions wasassessed by appearance of crystalline material by polarized lightmicroscopy (P.L.M.). Separate aliquots for each timepoint and storagecondition were made for every dispersion by placing 50 mg of material inopen glass vials. Vials were placed in appropriate temperaturecontrolled stability chambers which utilized saturated salt solutions tocontrol humidity (magnesium chloride for 33% RH, and sodium chloride for75% RH). Stability data for T=0 and T=1 month time point for thedispersions and conditions tested is shown in the table below.

12% K-15 12% K-90 12% K-30 20% K-30 30% K-30 HPLC HPLC HPLC HPLC HPLCPurity Appearance Purity Appearance Purity Appearance Purity AppearancePurity Appearance Conditions (%) by P.L.M. (%) by P.L.M. (%) by P.L.M.(%) by P.L.M. (%) by P.L.M. T = 0 97.10 amorphous 97.30 amorphous 99.50amorphous 99.60 amorphous 99.70 amorphous RT Stability time pointsamorphous 98.62 amorphous 99.15 amorphous 99.42 amorphous RT, 33% RHstopped at 2 weeks amorphous 98.71 amorphous 99.29 amorphous 99.40amorphous RT, 75% RH 97.09 amorphous 98.09 amorphous 98.89 amorphous99.24 amorphous 40° C. 98.57 amorphous 98.13 amorphous 98.87 amorphous99.34 amorphous 40° C., 75% 91.49 some 93.04 some 94.96 some 96.86 someRH crystalline crystalline crystalline crystalline material materialmaterial material visible visible visible visible

Example 18 Preparation of a Solid Dispersion [17-AAG in PEG6000 (5%w/w)]

To a solution of geldanamycin (20.0 g, 35.7 mmol, 1 eq.) in DCM (750 mL)was added allylamine (53 mL, 714 mmol, 20 eq) at room temperature andunder nitrogen atmosphere. The slurry was stirred at room temperaturefor 6 hours. The resulting purple solution was quenched with water (300mL) and acidified with 2N HCl (300 mL) to pH 3 and stirred for andadditional 30 min. The aqueous phase was extracted with DCM (300 mL) andthe combined organic layers washed with water (300 mL), dried overMgSO₄, filtered, and concentrated. The purple residue was dissolved intoacetone (300 mL) at 60° C. and heptanes (1.5 L) was added and theresulting mixture cooled to 5° C., filtered, and the solid washed withheptane (200 mL) to afford crude 17-AAG (18.15 g) after drying. Thepurple solid was dissolved in of acetone (306 mL) heated to 55-60° C.and n-heptane (1.2 L) was slowly added to form a slurry. The mixture wasmaintained at 55° C. for 30 minutes and cooled to room temperature. Thecrystalline material was collected and dried under vacuum for 48 hoursto afford 17-AAG as purple needles. (16.15 g, 28 mmol, 77% yield). >99%pure by HPLC monitored@254 nm) mp 210-212° C.

To 17-AAG (18 mg) was added PEG6000 (382 mg) and the solid mixture wasmelted using heat. The resulting waxy dispersion was analyzed by HPLCand by Cross Polar Microscopy.

Additional solid amorphous dispersions containing 17-AAG were prepared,the amorphous character was confirmed by polarized light microscopy andare summarized in the following table.

Composition/ TA Load Solvent Physical (%) Polymer Grade Method (EtOH)appearance Dissolution 95 PEG 1000 melt/fusion 2 PEG 1000 melt/fusion 2PEG 6000 melt/fusion slow dissolution, glassy material 3 PVP K-90 rotary 2 ml slow dissolution, evaporation glassy material 4 PVP K-90 rotary  2ml slow dissolution, evaporation glassy material 5 PEG 1000 melt/fusion5 PEG 6000 melt/fusion appears amorphous by PLM 17 PVP K-30 rotaryappears amorphous evaporation by PLM 33 PVP K-30 rotary appearsamorphous evaporation by PLM 25 PVP K-30 mechanical mix 30 PVP K-30rotary 35 ml appears amorphous evaporation by PLM 13 PVP K-30 rotary 10ml appears amorphous evaporation by PLM 5 PVP K-30 rotary 10 ml appearsamorphous evaporation by PLM 10 PVP K-30 rotary 10 ml appears amorphousevaporation by PLM 20 PVP K-30 rotary 10 ml appears amorphousevaporation by PLM 25 PVP K-30 rotary 10 ml appears amorphousevaporation by PLM 12 PVP K-30 rotary 100 ml  appears amorphousincreased evaporation by PLM solubility by in- vitro dissolution

Example 19 Preparation of 17-AG Amorphous Solid Dispersion Formulations[17-AG (20% load) in Various Polymers]

Method for Preparing Dispersions:

17-AG (2 g) was added to EtOH (1 L) in a 3 L one-necked flask. Thismixture was turned on the rotary evaporator at 60° C., ambient pressureand aggressive turning. An aliquot was taken and examined for signs ofcrystallization under the microscope after 1 hour. Polymer (8.2 g) wasadded and the mixture was concentrated via rotary evaporation and turnedat 60° C. for another hour. An aliquot was taken and examined for signsof crystallization using PLM after the hour. Vacuum was then applied andthe EtOH was removed over the course of 30 minutes to provide afoam-like material. The material was dried in vacuo over night. Theresulting brittle foam was then scraped from the sides and the materialwas pumped under high-vacuum for an additional 36 hours. The soliddispersion generated was then ground and sieved (No. 50 sieve) to aparticle size of 300 microns.

Example 20 General Techniques Utilized to Characterize AmorphousMaterials

Visual Polarized Light Microscopy (PLM): A check (“√”) indicates novisual signs of crystalline material and no birefringence under crosspolarized light when examining the solid state material or whendissolved in water.

DSC: A check (“√”) indicates identification of a glass transitiontemperature (Tg), no apparent crystal endotherm. Exemplary micrographsare shown in FIG. 44 and FIG. 45 (amorphous dispersion visualized underPLM).

XRPD: A check indicates no crystalline signature.

In vitro Dissolution: A check indicates a supersaturated level of 17-AGof at least 0.4 mg/ml (50% of 12% 17-AG K-30).

HNMR: A check (“√”) indicates a spectrum consistent with the expectedstructure and one that shows<5% residual solvents.

LCUV: A check indicates>95% purity.

Stability: A check a check (“√”) indicates TG-DSC>40° C. above RT, >1month stability@room temperature by LCUV, DSC, microscopy.

Characteristics of Amorphous 17-AG Solid Dispersions in Various Polymers

17-AG 17-AG in 17-AG in 17-AG in 17-AG in in PVP EUDRAGIT PLASDONE HPMCPHPMCAS Visual √ √ √ √ √ (PLM)- solid Visual- √ √ √ √ √ dissolved inwater ¹H NMR √ √ √ √ √ TG-DSC √ √ √ √ √ XRPD √ √ √ √ √ (@ 33% load)Purity √ √ √ √ √

Preparation of Samples for in-vitro Dissolution: a Series ofScintillation Vials was used to prepare samples according to thefollowing table, containing 17-AG (20% load) polymer dispersion (50.0mg). To each vial was added simulated intestinal fluid (5 mL) and eachwas shaken at 37° C. At 5-, 15-, 30-, 60-, 90,- 120-min, 4-hour, 8-hourand overnight time-points, aliquots (300 uL) of each suspension weredrawn and filtered via polypropylene filter (0.45 micron) into MeOH (750uL). The samples were then diluted in MeOH and tested using UV methodfor solution concentration. The results of each sample are summarized inthe following tables.

Dissolution Experiment Setup

Dis- Polymer persion 17- Target from Solution weight AG Conc dispersionPolymer volume Sample (mg) (mg) (mg/mL) (mg) (%) (mL) 17-AG in 50.0 10 240.0 0.80 5 PVP 17-AG in 50.0 10 2 40.0 0.80 5 HPMCP 17-AG in 50.0 10 240.0 0.80 5 HPMCAS 17-AG in 50.0 10 2 40.0 0.80 5 PLASDONE S-630 17-AGin 50.0 10 2 40.0 0.80 5 EUDRAGIT L100

The following are data collected from an in vitro dissolution studydemonstrating that supersaturated levels of 17-AG are achieved, relativeto equilibrium solubility, when crystallization inhibitors are used toprepare solid dispersion formulations (resulting in supersaturatedlevels of 17-AG when measured by dissolution in vitro). Suchformulations can be prepared utilizing various types of crystallizationinhibitors or polymers (other than PVP). Exemplary crystallizationinhibitors utilized are HPMCP, HPMCAS, PLASDONE S-630 (a vinylpyrrolidone and vinyl acetate copolymer) and EUDRAGIT L100.

Dissolution Summary Results

Supersaturated Equilibrium Concentration of 17-AG solubility of fromVarious Polymer 17-AG 17-AG in dispersions in Load SIF @ 37° C. SIF @37° C. (mg/mL) Polymer (w/w %) (mg/mL) (t = 5 min) PVP K30 20 0.0040.459 mg/mL (100X)* HPMCP 20 0.004 0.250 mg/mL HP-55  (60X)* HPMCAS 200.004 0.283 mg/mL HG  (70X)* PLASDONE 20 0.004 0.522 mg/mL S-630 (130X)*EUDRAGIT 20 0.004 0.420 mg/mL L100 (100X)* *Fold increase relative to17-AG equilibrium solubility

FIG. 13 shows the in vitro dissolution of 17-AG dispersions made usingthe above various crystallization inhibitors.

Amorphous dispersions can be made using other geldanamycin analogs aswell. To similarly demonstrate that compounds other than 17-AG benefitfrom the addition of a crystallization inhibitor, Example 21 below is anin-vitro dissolution study demonstrating that supersaturatedconcentrations of geldanamycin analogs can be achieved when acrystallization inhibitor is added to a substantially amorphous soliddispersion formulation, which may also result in improved in vivobioavailability as seen for the analog 17-AG. Such formulation can beprepared utilizing various geldanamycin analogs. Particular analogsutilized are 17-benzyl-AG, 17-fluoroethyl-AG, 17-cyclopropylmethyl-AG,17-acetyl-AG, 17-azetidinyl-G, 11-acetyl-17-AG and 11-oxo-17-AG. Theresults of each analog are summarized in the below tables in Example 21.

Example 21 Preparation of Amorphous Solid Dispersion Formulations UsingVarious Geldanamycin Analogs [17-AG (12% load) plus PVP]

A mixture of a geldanamycin compound and solvent is heated and stirredvigorously. Aliquots are periodically taken and examined via polarizedlight microscopy to determine complete dissolution of the crystallinematerial. Upon complete dissolution, the crystallization inhibitor isslowly added to the solution. The mixture is stirred vigorously withheat, and aliquots are taken and examined via microscopy to ensurecomplete dissolution of the components. Alternatively, the order ofaddition can be changed so that the polymer is used as a co-solvent,e.g., the compound is added to a pre-mixed solution of the polymer andthe appropriate ratio or combination of solvents. The dispersions werecharacterized as described above. Characteristics of Amorphous SolidDispersions of Geldanamycin Analogs

17- 11- 17- 17- cyclopropyl 17- 17- 11- oxo- benzyl- fluoroethyl-methyl- acetyl- azetidinyl- acetyl- 17- 17- AG AG AG AG AG 17-AG AG AGVisual-solid √ √ √ √ √ √ √ √ Visual- √ √ √ √ √ √ √ √ solubility ¹H NMR √√ √ √ √ √ √ √ DSC-T_(g) √ √ √ √ √ √ √ √ DSC √ √ √ √ √ √ √ √crystallinity Purity √ √ √ √ √ √ √ √

Using a procedure similar to Example 20, a senes of scintillation vialswas prepared according to the table below, except that analogs of 17-AG(12% load) plus PVP K-30 (83.3 mg) were used accordingly.

Dissolution Experiment Setup

Dispersion Analog Target PVP from Solution weight amount Conc dispersionvolume Sample (mg) (mg) (mg/mL) (mg) PVP (%) (mL) 17-AG 83.3 10 2 731.47 5 17-benzyl-AG 83.3 10 2 73 1.47 5 17-fluoroethyl-AG 83.3 10 2 731.47 5 17- 83.3 10 2 73 1.47 5 cyclopropylmethyl- AG 17-acetyl-AG 83.310 2 73 1.47 5 17-azetidinyl-G 83.3 10 2 73 1.47 5 11-acetyl-17-AG 83.310 2 73 1.47 5 11-oxo-17-AG 83.3 10 2 73 1.47 5

Summarized below are the results from an in-vitro dissolution study fordispersions containing amorphous material made using a wide variety ofansamycin compound analogs. The data demonstrate that supersaturatedlevels of a variety of ansamycin compounds can be achieved. Thedissolution profile of amorphous dispersions of the series of ansamycinanalogs generated from PVP utilizing rotary evaporation is found in FIG.15.

Geldanamycin Analog Summary Results

Supersaturated concentration of Eq. Solubility analog/polymerdispersions of active in SIF at 37° C. Analog (mg/mL) (mg/ml) (t = 5min) 17-AG 0.004  0.712 mg/ml (200x) 17-benzyl-AG 0.015  0.165 mg/ml(10x) 17-fluoroethyl-AG 0.040 0.234 mg/ml (5x) 17-cyclopropylmethyl-AG0.034 0.0573 mg/ml (2x)  17-acetyl-AG 0.350    1.568 mg/ml (≧5x)17-azetidinyl-G 0.140 0.167 mg/ml (0x) 11-acetyl-17-AG 0.088 0.167 mg/ml(2x) 11-oxo-17-AG 0.330 0.395 mg/ml (0x) *Fold increase relative toequilibrium solubility

Example 22 below demonstrates that supersaturated solutions of 17-AGgenerated from substantially amorphous solid dispersion formulationsthat have been “spiked” with various amounts of crystalline material(0.01%, 0.1% 1% and 10%, with 0% as a comparator) demonstrate alteredprecipitation kinetics of 17-AG. Adding various amounts of crystallinematerial to the amorphous dispersions of 17-AG affects the stability ofthe corresponding supersaturated solution. Increasing amounts ofcrystalline material present in the supersaturated solutions increasesthe rate of 17-AG nucleation and precipitation. The in vitroexperimental dissolution protocol of each spiked formulation issummarized in the tables below. A representative DSC of 20% 17-AG inPVP-K30 is found in FIG. 16. Additional data illustrating variousamounts of Spiking with Form I are found in FIGS. 16, 17, 18, 19, 20,21, 22 and 23. Spiking with Forms II and III results in similarfindings. As shown in these Figures, there is a measurable differencebetween the formulations containing “0%” crystalline material, and theformulations spiked with 1% crystalline material, demonstrating that theformulation designated “0%” contains less than 1% crystalline material.

Example 22 Dissolution Study Using 17-AG in Spiking Experiment

To 1 dram vials containing a solid dispersion of 17-AG (20% load) plusPVP K30 were added various amount of 17-AG Form I to provide a totalmass of 500 mg and crystalline range from 0.01 to 10%. To ensurehomogenous mixtures of dispersion and crystalline 17-AG, the mixtureswere ground with mortar and pestle, sieved through a No. 50 screen (300um) and mixed for 5 minutes using a Turbula Mixer. The amount ofcrystalline material was examined by microscopy. Substantially amorphousmaterial had no visible crystalline material and no birefringence undercross polarized light with dry material and upon introduction of water.

Amount of 17- Total % of Figure dispersion AG added material crystallineReference Sample (mg) (mg) (mg) material No.   0% Spiked 500.0 0.0 500.00 44(A) and 44(B) 0.01% Spiked 999.9 0.1 1000.0 0.01 44(C)  0.1% Spiked499.5 0.5 500.0 0.1 44(D)   1% Spiked 495.0 5.0 500.0 1 44(E)   10%Spiked 450.0 50.0 500.0 10 —

To a scintillation vial containing the prepared spiked soliddispersions, (prepared according to the schedule in the table below) wasadded simulated intestinal fluid (5 mL); the vials were then shaken at37° C. At 5-, 15-, 30-, 60-, 90,- 120-min, 4-hour, 8-hour and overnighttime-points, aliquots (300 uL) of each sample were drawn and filteredvia a polypropylene filter (0.45 micron) into MeOH (750 uL). The sampleswere then diluted in MeOH and tested using UV method for solutionconcentration. The results are summarized in the following table.

Additionally, FIG. 44(B) and FIG. 44(C) show a visual difference betweena non-spiked dispersion and a dispersion spiked with 0.01% crystallinematerial, demonstrating that the non-spiked dispersion contains lessthan 0.01% crystalline material.

Spiking Experiment Dissolution Study-Sample Preparation

Dispersion Sample PVP from Total Solution weight amount Conc dispersionPVP add material % volume Sample (mg) (mg) (mg/mL) (mg) back (mg) (mg)PVP (mL)   0% Spiked 50.0 10 2 40 0 50.0 0.80 5 0.01% Spiked 50.0 10 240 0 50.0 0.80 5  0.1% Spiked 50.0 10 2 40 0 50.0 0.80 5   1% Spiked48.1 10 2 38.1 1.9 50.0 0.80 5   10% Spiked 35.7 10 2 25.7 14.3 50.00.80 5

Example 23 Control of Release Rate for 17-AG/PVP Dispersion Solid DoseForms

To demonstrate that the control of the release rate of 17-AG from 17-AGplus PVP dispersions is feasible, tablets and capsules were made ofvarying composition/excipients from a 20% 17-AG plus PVP K30 dispersionwhich have immediate, extended, and slow release rates as measured byin-vitro dissolution. As such, controlling the dissolution rate, inExample 24 below, of solid dose forms of 17-AG amorphous dispersionscould provide a means of controlling the degree of supersaturation ofdissolved 17-AG when dosed in-vivo. The composition of the tablets andcapsule are described in the following table.

Dose Name Form Composition 25 mg capsule 100% 20% IPI-493 PVP rotaryevaporation DPI, capsule size 4 HPMC capsule 50 mg capsule 100% 20%IPI-493 PVP rotary evaporation DPI, capsule size 2 HPMC capsule Tablet 1tablet 79.5% 20% IPI-493 PVP spray dried DPI, 20% Explotab, 0.5%magnesium stearate, hard compression Tablet 2 tablet 89.5% 20% IPI-493PVP spray dried DPI, 10% Explotab, 0.5% magnesium stearate, hardcompression Tablet 3 tablet 95.5% 20% IPI-493 PVP spray dried DPI,, 4%Explotab, 0.5% magnesium stearate, soft compression Tablet 4 tablet95.5% 20% IPI-493 PVP spray dried DPI, 4% Explotab, 0.5% magnesiumstearate, hard compression

Rate of release and level of dissolved 17-AG was measured by dissolvingthe tested tablet or capsule in 500 ml SIF pH 6.8 and stirred in adissolution apparatus (Paddle speed 150 RPM, 37° C.) until completelydissolved. Aliquots of the tablet/capsule-SIF solution were removed at15, 30, 90, 120, 180, & 240 minute timepoints. Sample aliquots werefiltered (0.45 uM PVDF), diluted in SIF/MeOH and measured in triplicateon UV spectrometer.

The dissolution profile of 17-AG from the different tablets and capsulesis shown in the graph in FIG. 24. As demonstrated in the results, it ispossible to produce tablets and capsules with different in-vitro releaserates (immediate, extended, & slow).

Example 24 Effect of Crystallization Inhibitors on Supersaturation

Crystallization inhibitors such as PVP (polyvinylpyrrolidone, Povidone)can improve the solubility of compounds by preventing crystallization.The effect of PVP on the amount of solvated 17-AG was investigated bymeasuring levels of 17-AG in SIF pH 6.8 (simulated intestinal fluid)solutions with increasing amounts of PVP at various time pointsfollowing the addition of specific amounts of 17-AG, either incrystalline form or in DMSO solutions to supersaturated levels.

Equilibrium solubility of crystalline 17-AG was measured at specifictime points by addition of crystalline 17-AG to SIF pH 6.8 containing0%, 0.5%, 1%, 2.5%, & 5% PVP (w/v) to a concentration of 5 mg/ml.Solutions were placed at 37° C., and sample aliquots were removed at 24,48, 72, & 96 hours. Sample aliquots were filtered (0.45 uM PVDF),diluted in SIF/MeOH and measured in triplicate on UV spectrometer.

Supersaturated levels of 17-AG was measured by adding 17-AG DMSO stocksolutions (10, 50, 100, & 200 mg/ml) at a 1:100 dilution to SIF pH 6.8solutions containing 0%, 0.5%, 1%, 2.5%, & 5% PVP (w/v). This resultedin solutions with final 17-AG concentrations of 0.1, 0.5, 1, & 2 mg/mlrespectively for each of the SIF pH 6.8 PVP solutions. Solutions werestirred in a dissolution apparatus (Paddle speed 150 RPM, 37° C.), andaliquots of each solution were removed at 15, 30, 60, 120, 240, 360, &1320 minute time points. Sample aliquots were filtered (0.45 uM PVDF),diluted in SIF/MeOH and measured in triplicate on UV spectrometer.Representative data are found in FIG. 25 and FIG. 26.

Summary of 17-AG Solubility Results (Average Values)

Crystalline 17-AG SIF pH 6.8 Equilibrium 17-AG Supersaturated solution-%PVP Solubility (mg/ml) Concentration (mg/ml)   0% PVP 0.0041 0.5 0.5%PVP 0.0059 0.6 1.0% PVP 0.0065 0.7 2.5% PVP 0.0087 — 5.0% PVP 0.0112 0.9

As shown above, PVP improves the levels of solvated 17-AG. Theequilibrium solubility of crystalline 17-AG increases almost 3-fold from0.0041 mg/ml in SIF pH 6.8 with 0% PVP to 0.0112 mg/ml in SIF pH 6.8with 5% PVP. In addition, the degree of supersaturation of 17-AGincreases almost 2-fold from 0.5 mg/ml at 0% PVP to 0.9 mg/ml at 5% PVP.

In addition, PVP enhances the stability of the supersaturated solutionsby prolonging the duration of the supersaturated state. In SIF withoutPVP, 17-AG starts to precipitate and come out of solution atapproximately 120 minutes. In SIF solutions with PVP, the 17-AGsupersaturated state can be prolonged beyond 120 minutes to almost 240minutes. The rate of 17-AG precipitation is also attenuated by PVP in aconcentration dependent manner, i.e. the higher % PVP, the slower therate of 17-AG precipitation from the supersaturated state. However, itappears that this crystallization rate inhibitory effect of PVP can beovercome by precipitation of 17-AG from very high supersaturated levelsas seen in the result of the 1.0 mg/ml solution in 5% PVP which achievesa supersaturated state of 0.9 mg/ml; however starts to precipitate outat 120 minutes. Representative data are shown in FIG. 27.

Example 25 Comparison of in vivo Exposure in Beagle Dogs of 17-AG PVPDispersions

The effects of varying compound load, PVP grade and particle size in17-AG+PVP dispersions on oral bioavailability was investigated by dosingbeagle dogs and measuring 17-AG levels in blood plasma at various timepoints following a single oral capsule dose.

The following 17-AG+PVP dispersions were made utilizing rotaryevaporation and characterized for purity, residual solvent level, andamorphous content as described.

17-AG PVP Grade (% Load) K15 K30 K90 12 12% 17-AG K15 12% 17-AG K30 12%17-AG K90 20 20% 17-AG K30 30 30% 17-AG K30

Each 17-AG+PVP dispersion was filled into HPMC capsules and dosed intodogs at a level of 15 mg/kg. Blood was collected pre-dose, 15, 30minutes, 1, 2, 4, 8, and 24 hours post dose into tubes containing sodiumheparin. Collected blood samples were immediately placed on wet ice andrefrigerated centrifuge for isolation of plasma within 30 min ofcollection. Isolated plasma was saved in labeled screw cap freezer vialsor eppendorf tubes and stored frozen (−70° C.) until analyzed for plasma17-AG levels. The design of the study is as follows. 2 groups of dogswere utilized for dosing, 2 males & 2 females per group, with a weekwashout in between each dose. For specifics of the animal dosingprotocol, please refer to PCRS protocol No. INF-0704.

Dog Group Dose 1 Dose 2 Dose 3 Dose 4 17-AG A 12% 17-AG PVP 20% 17-AGPVP 30% 17-AG PVP 20% 17-AG PVP Load K30 @15 mg/kg K30 @15 mg/kg K30 @15mg/kg K30 <50 uM @10 mg/kg PVP B 12% 17-AG PVP 12% 17-AG PVP 12% 17-AGPVP 20% 17-AG PVP Grade K30 @15 mg/kg K15 @15 mg/kg K90 @15 mg/kgK30 >800 uM @10 mg/kgAfter analysis of 17-AG plasma levels following dosing, there was not asignificant effect on exposure due to either 17-AG load or PVP grade;however, there are exposure trends. For 17-AG load, C_(max) and AUCdecreased with increasing 17-AG load (12%>20%>30% 17-AG). For PVP grade,C_(max) and AUC were highest in PVP K30, followed by PVP K15, followedby PVP K90. No trends or affects on C_(max) or AUC could be seen due tochanges in particle size, i.e. exposure is robust across a large rangeof particle sizes. Overall, there were no consistent differences seen inthe exposure data due to sex for the variables tested. Any variabilityobserved in the exposure is most likely due to animal specificdifferences or in-life observations (i.e. dosing issues, emesis).

Summary of the oral bioavailability results (average values) reflectingvarious load levels are found in FIG. 28 a and FIG. 29 a, andaccompanying summary data tables in FIG. 28 b and FIG. 29 b. Summary ofthe oral bioavailability results (average values) reflecting various PVPgrades are found in FIG. 41 a and FIG. 42 a, and accompanying summarydata tables in FIG. 41 b and FIG. 42 b. Summary of the oralbioavailability results (average values) reflecting various particlessizes of 20% 17-AG and PVP K-30 are found in FIG. 40 a, and accompanyingsummary data table in FIG. 40 b.

Example 26 In Vivo Exposure of Various Oral Formulations of 17-AG inBeagle Dogs

The oral bioavailability of various formulations of the compound 17-AGwas investigated by making various oral formulations, dosing beagle dogsand measuring 17-AG levels in blood plasma at various time pointsfollowing a single oral dose. In addition, the effect of PVP onenhancing exposure was investigated by its addition to many of thetested formulations.

In brief, different crystalline, solvated, and amorphous forms of 17-AGwere dosed either as filled capsules or as suspensions. In addition,various 17-AG solutions utilizing a range of different organic, anionic,& non-ionic components were dosed by oral gavage. The different oralformulations tested are listed in the following table.

Dose of 17- Figure No. Formulation/Dose 17-AG dose form AG (mg)Reference 20% 17-AG + PVP K30 rotary evaporation amorphous dispersion in50 14a dispersion capsule 20% 17-AG + PVP K30 spray dried dispersionamorphous dispersion in 50 14b capsule 17-AG crystalline + lactosecrystalline solid in capsule 50 — 17-AG crystalline + PVP crystallinesolid in capsule 50 — 17-AG Ethyl acetate solvate + lactose crystallinesolid in capsule 50 30a and 30c 17-AG Ethyl acetate solvate + PVPcrystalline solid in capsule 50 30b and 30c Amorphous 17-AG + lactoseamorphous solid in capsule 50 31a and 31c Amorphous 17-AG + PVPamorphous solid in capsule 50 31b and 31c 2 mg/ml 17-AG in 85% PG, 10%DMSO, 5% solution 50 32 EtOH 2 mg/ml 17-AG in 85% PG, 10% PVP, 5%solution 50 33 EtOH 2 mg/ml 17-AG in 20% PGHS, 5% DMSO in solution 50 34NS 2 mg/ml 17-AG in 20% PGHS, 5% DMSO, solution 50 35 10% PVP in NS 2mg/ml 17-AG in 2% Tween-80, 5% DMSO in solution 50 36 SWFI 2 mg/ml 17-AGin 2% Tween-80, 5% DMSO, solution 50 37 10% PVP in SWFI 2 mg/ml 17-AG in0.17% SLS, 5% DMSO in solution 50 — SWFI 2 mg/ml 17-AG in 0.17% SLS, 5%DMSO, solution 50 — 10% PVP in SWFI 2 mg/ml 17-AG in 10% PGHS, 2.5%DMSO, emulsion 50 — 5% Tween-80, 50% olive oil in NS 2 mg/ml 17-AG in10% PGHS, 2.5% DMSO, emulsion 50 — 5% Tween-80, 5% PVP, 50% olive oil inNS 12% 17-AG HPMC-AS rotary evaporation amorphous dispersion in 50 —dispersion capsule 12% 17-AG EUDRAGIT L100 rotary evaporation amorphousdispersion in 50 — dispersion capsule 2% 17-AG nano-suspension in 2%Tween-80 crystalline solid in suspension 50 — 2% 17-AG nano-suspensionin 2% Tween-80, crystalline solid in suspension 50 — 10% PVP 2% 17-AGsuspension in 1% crystalline solid in suspension 15 mg/kg —Carboxymethylcellulose

The crystalline, solvated, and amorphous forms of 17-AG were made aspreviously described and blended with either anhydrous lactose or PVP ina Turbula blender for 15 minutes.

Solutions were made by adding the formulation components to the desiredpercentage by weight and either adding 17-AG to the desiredconcentration of 2 mg/ml until dissolved or by adding a stock solutionof 40 mg/ml 17-AG in DMSO and diluting 1:20 to achieve the finalconcentration of 2 mg/ml. All solutions were clear and purple in colorwithout any evidence of precipitate.

The suspension in carboxymethylcellulose was made by levigatingcrystalline 17-AG with glycerol in a mortar and pestle followed byhomogenization in the 1% carboxymethylcellulose solution in a high speedhomogenizer for 10 minutes. Homogeneity of the suspension was checked bymicroscopy. FIG. 46(A) is an exemplary photo of a suspension of 17-AG,in 1% carboxymethylcellulose.

The emulsions were made by making a 4 mg/ml solution of 17-AG in either10% PGHS/2.5% DMSO/5% Tween-80 or 10% PGHS/2.5% DMSO/5% Tween-80/5% PVP,combining the solution 1 to 1 with olive oil followed by mixing in ahigh speed homogenizer for 15 minutes. Confirmation of the emulsion wasperformed by microscopy. FIG. 46(B) is an exemplary photo of an emulsionof 17-AG, in 10% PGHS, 2.5% DMSO, 5% Tween-80, 50% olive oil, in NS.

The nanosuspension was made by levigating crystalline 17-AG by highshear in a microfluidizer (Microfluidics Corp, Model: M-110L) for 10-20minutes in Tween-80. Mean particle size (d50: 300-400 nM) was measuredby laser light diffraction (Malvern Corp, Mastersizer 2000).

All dosed formulations had at least 2 hours physical stability at roomtemperature.

Blood was collected pre-dose, 15, 30 minutes, 1, 2, 4, and 8 hourspost-dose into tubes containing sodium heparin. Collected blood sampleswere immediately placed on wet ice and refrigerated centrifuge forisolation of plasma within 30 min of collection. Isolated plasma wassaved in labeled screw cap freezer vials or eppendorf tubes and storedfrozen (−70° C.) until analyzed for plasma 17-AG levels. The design ofthe study is as follows. Two groups of dogs, each group consisting of 3females were utilized for dosing, with a week washout in between eachdose.

Group A Group B Study 1 20% 17-AG PVP K30, rotary evaporation Study 217-AG + Lactose Study 3 17-AG + PVP K30 Study 4 17-AG EtOAc Solvate +Lactose Study 5 20% 17-AG PVP K30, spray dried 17-AG EtOAc Solvate + PVPdispersion Study 6 Amorphous 17-AG + Lactose Organic PG/EtOH + DMSOsolution Study 7 Amorphous 17-AG + PVP Organic PG/EtOH + PVP solutionStudy 8 PEG-HS solution Non-ionic Tween 80 solution Study 9 PEG-HS + PVPsolution Non-ionic Tween 80 + PVP solution Study 10 Anionic mic SLSsolution Oil Emulsion Study 11 Anionic mic SLS + PVP solution OilEmulsion + PVP Study 12 20% 17-AG HPMC-AS dispersion Study 13 20% 17-AGEUDRAGIT L100 dispersion Study 14 Nano suspension Study 15 Nanosuspension + PVP

For the solid formulations, PVP does not appear to enhance exposure forthe physical mixes of crystalline 17-AG and PVP to a level where 17-AGcan be detected. However, there was low exposure after dosing the EtOAcSolvate of 17-AG, and significant exposure after dosing amorphous 17-AGor amorphous dispersions of 17-AG. Moreover, the inclusion of PVP tothese formulations appears to improve the exposure profiles of the EtOAcSolvate and amorphous 17-AG. This result is consistent with the in-vitrodissolution experiments demonstrating the ability of PVP to enhance17-AG solubility and stabilize supersaturated solutions of 17-AG. Methodof manufacture does not appear to have an effect since the exposure fromdosing either solvent evaporated or spray dried 20% 17-AG/PVP amorphousdispersion is the same. Also consistent with the results from the17-AG/PVP dispersions are the in-vivo results from dosing soliddispersions of 17-AG with other crystallization inhibitors, HPMC-AS andEUDRAGIT L100. The improved bioavailability of 17-AG from theseformulations as compared to dosing crystalline 17-AG demonstrates theutility of utilizing crystallization inhibitors in amorphous dispersionwith geldanamycin analogs.

For the liquid (solution, emulsion) formulations, there was asignificant exposure for all of the formulations dosed. Inclusion of PVPin the formulation did not appear to have the same significant effect inthe solution doses as it did in the solid dose exposure results. Thiscould be due to the fact that all of the solutions were dosed at arelatively low concentration (2 mg/ml) and small volume (25 ml), and hadgood physical stability. The consistently high exposure results for allof the solution doses would suggest that 17-AG is being readily absorbedbefore PVP could demonstrate its ability to stabilize the solution.

For the suspension and nano-suspension formulations, there was little orno measurable level of 17-AG following dosing, either with or withoutthe crystallization inhibitor PVP.

In total, these results demonstrate the ability to dose 17-AG utilizinga wide range of oral formulations. Any variability observed in theexposure is most likely due to animal specific differences or in-lifeobservations (i.e. dosing issues, emesis). Exemplary graphs of theexposure data for specific doses are found in the Figure No. Referencesas specified in the table below. A summary of the oral bioavailabilityresults (average values) is found in the following table:

AUC INF Figure Half T_(max) C_(max) (ng * hr/ No. Formulation/Dose Life(h) (h) (ng/ml) ml) Reference 20% 17-AG + PVP K30 rotary evaporation 1.51.8 1176.2 3153.0 14a dispersion 20% 17-AG + PVP K30 spray drieddispersion 1.2 1.7 1450.0 3011.3 14b 17-AG crystalline + lactose C.N.E.C.N.E. BLQ C.N.E. 17-AG crystalline + PVP C.N.E. C.N.E. BLQ C.N.E. 17-AGEthyl acetate solvate + lactose 2.7 0.7 98.1 239.0 30a and 30c 17-AGEthyl acetate solvate + PVP 3.2 1.0 124.4 322.2 30b and 30c Amorphous17-AG + lactose 1.5 1.0 942.3 1854.4 31a and 31c Amorphous 17-AG + PVP1.3 1.2 1141.3 2594.2 31b and 31c 2 mg/ml 17-AG in 85% PG, 10% DMSO, 5%1.3 2.0 2170.0 4937.1 32 EtOH solution 2 mg/ml 17-AG in 85% PG, 10% PVP,5% 1.4 1.7 1696.7 4454.4 33 EtOH solution 2 mg/ml 17-AG + 20% PGHS, 5%DMSO in 1.4 1.2 908.3 2894.3 34 NS solution 2 mg/ml 17-AG + 20% PGHS, 5%DMSO, 1.5 1.2 761.3 2512.8 35 10% PVP in NS solution 2 mg/ml 17-AG in 2%Tween-80, 5% DMSO in 1.5 0.8 1393.3 3915.6 36 SWFI solution 2 mg/ml17-AG in 2% Tween-80, 5% DMSO, 1.5 0.6 871.3 2116.6 37 10% PVP in SWFIsolution 2 mg/ml 17-AG in 0.17% SLS, 5% DMSO in 1.2 1.0 2362 6090 — SWFIsolution 2 mg/ml 17-AG in 0.17% SLS, 5% DMSO, 1.4 0.4 1788 3300 — 10%PVP in SWFI solution 2 mg/ml 17-AG in 10% PGHS, 2.5% DMSO, 1.3 0.9 10112923 — 5% Tween-80, 50% olive oil in NS emulsion 2 mg/ml 17-AG in 10%PGHS, 2.5% DMSO, 1.6 0.5 765 2394 — 5% Tween-80, 5% PVP, 50% olive oilin NS emulsion 12% 17-AG HPMC-AS rotary evaporation 1.19 2 2133 6901 —dispersion 12% 17-AG Eudragit L100 rotary evaporation 1.6 0.5 765 2394 —dispersion 2% 17-AG nano-suspension in 2% Tween-80 C.N.E. 0.25 5.27C.N.E. — 2% 17-AG nano-suspension in 2% Tween-80, C.N.E. 0.50 8.50C.N.E. — 10% PVP 2% 17-AG suspension in 1% C.N.E. C.N.E. BLQ C.N.E. —Carboxymethylcellulose CNE = cannot estimate.

Following Example 27 illustrates that 17-AG as a solution reduces tumorgrowth in a mouse xenograph model. Representative data are found in FIG.47 and FIG. 48.

Example 27 In-Vivo Efficacy of 17-AG

In-vivo efficacy of 17-AG was demonstrated by conducting in 2 mousexenograph studies utilizing mouse xenograph tumor models of clientproteins dependent upon HSP90.

In the first study, H1975, a non-small cell lung cancer cell line whichcontains L858R and T790M mutations in EGFR, a client protein for HSP90was utilized. 5-6 week old Nu/Nu mice were implanted with 10×10e6H1975cells. Dosing commenced after implanted cells reached ˜150 mm3. Dosingwas by oral gavage and the dosing schedule was every other day withvehicle (20% PGHS, 5% DMSO, 75% NS), and 75 mg/kg, and 100 mg/kg 17-AGin vehicle. After dosing, ˜35% and ˜70% reduction in tumor volume wasseen in the dosing arms as compared to vehicle treated animalsdemonstrating efficacy of dosing 17-AG in a xenograph tumor modeldependent upon an HSP90 client protein.

In the second study, H1650 lung adenocarcinoma cell line which containsa mutant form of EGFR (Del E746-A750) was utilized. 5-6 week old Nu/Numice were implanted with 10×10e6H1650 cells. Dosing commenced afterimplanted cells reached ˜100 mm3. Dosing was by oral gavage and thedosing schedule was every day with vehicle (15% PVP, 5% EtOH, 80% PG),and 50 mg/kg, 75 mg/kg, and 100 mg/kg 17-AG in vehicle. After dosing,62% maximum reduction in tumor volume was seen in the dosing arms ascompared to vehicle treated animals demonstrating efficacy of dosing17-AG in a xenograph tumor model dependent upon on an HSP90 clientprotein.

Other embodiments included herein are provided in the following claims.

1. A pharmaceutical composition comprising a substantially amorphousbenzoquinone ansamycin compound formulated for oral administration as amolecular dispersion with a crystallization inhibitor to provide an areaunder the curve (AUC) circulatory bioavailability of at least 1000ng·hr/ml wherein the compound is 17-amino-geldanamycin (17-AG).
 2. Acomposition comprising substantially amorphous 17-amino-geldanamycin(17-AG) formulated for oral delivery to a subject to provide an areaunder the curve (AUC) circulatory bioavailability of at least 1000ng·hr/ml.
 3. The composition according to claim 2 wherein the17-amino-geldanamycin (17-AG) is formulated for oral delivery at a doseof at least 5 mg.
 4. The composition according to claim 2 wherein the17-amino-geldanamycin (17-AG) is formulated for oral delivery at a doseof at least 25 mg.
 5. The composition according to claim 2 wherein the17-amino-geldanamycin (17-AG) is formulated for oral delivery at a doseof at least 50 mg.
 6. The composition according to claim 2 wherein the17-amino-geldanamycin (17-AG) is formulated for oral delivery at a doseof at least 125 mg.
 7. The composition according to claim 2 wherein the17-amino-geldanamycin (17-AG) is formulated for oral delivery at a doseof at least 250 mg.
 8. A composition comprising substantially amorphous17-amino-geldanamycin (17-AG) and at least about 10% by weight of acrystallization inhibitor.
 9. The composition according to claim 8,wherein the composition contains at least about 25% by weight of thecrystallization inhibitor.
 10. The composition according to claim 8,wherein the composition contains at least about 50% by weight of thecrystallization inhibitor.
 11. The composition according to claim 8,wherein the composition contains at least about 75% by weight of thecrystallization inhibitor.
 12. The composition according to claim 8,wherein the crystallization inhibitor is polyvinylpyrrolidone (PVP). 13.A pharmaceutical composition comprising substantially amorphous17-amino-geldanamycin (17-AG) or a pharmaceutically acceptable saltthereof, wherein the composition is present as a molecular dispersion.14. The composition according to claim 1 wherein the crystallizationinhibitor is selected from polyvinylpyrrolidone; crospovidone; gums; acellulose derivative which is selected from hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate, hydroxypropylcellulose, ethyl cellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, and sodium carboxymethylcellulose; dextran; acacia; homo- and copolymers of vinyllactam, andmixtures thereof; cyclodextrins; gelatins; hypromellose phthalate;sugars; polyhydric alcohols; polyethylene glycol; polyethyleneglycol-hydroxystearate; polyethylene oxides; polyoxyethylene; polyvinylalcohol; propylene glycol; sodium lauryl sulfate (SLS); Tween; andcombinations thereof.
 15. A pharmaceutical composition comprisingsubstantially amorphous 17-amino-geldanamycin (17-AG) or apharmaceutically acceptable salt thereof wherein the composition furthercomprises a polyvinylpyrrolidone.
 16. The composition according to claim15, wherein the polyvinylpyrrolidone is selected from homo- andco-polymers of polyvinylpyrrolidone; and homo- and co-polymers ofN-vinylpyrrolidone.
 17. The composition according to claim 1, wherein acrystallization inhibitor is present in an amount of at least about 5%,10%, 15%, or 25% (w/w), based on the total weight of the composition.18. The composition according to claim 17, wherein the amount is atleast about 10% (w/w), based on the total weight of the composition. 19.The composition according to claim 17, wherein the amount is at leastabout 15% (w/w), based on the total weight of the composition.
 20. Thecomposition according to claim 17, wherein the amount is at least about25% (w/w), based on the total weight of the composition.
 21. Thecomposition according to claim 13, wherein the molecular dispersionresults from: (a) milling; (b) extrusion; (c) melt processes; (d)solvent modified fusion; (e) solvent processes; or (f) non-solventprecipitation.
 22. The composition according to claim 21, wherein thesolvent processes are selected from lyophilization, rotary evaporation,spray coating and spray-drying.